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PubMed1978

Spin membrane immunoassay: simplicity and specificity.

The many disadvantages of radioimmunoassay (RIA) have stimulated attempts to develop non-radioactive assays. These include spin immunoassay (SIA), which is simple, specific, and requires no separation procedures but is much less sensitive than RIA. The membrane immunoassay (MIA) described here is more sensitive than the SIA. Serum is prepared as for RIA. The MIA employs liposomes sensitized with epsilon-dinitrophenylated aminocaproyl phosphatidylethanolamine. It records liposome lysis induced by specific anti-Dnp antibodies, and complement which is monitored by the release of trapped spin labels (N-(2,2,6,6-tetramethylpiperidinyl-1-oxyl)-choline chloride). The sensitized liposomes are stable and give reproducible results for up to 4 weeks. The system's sensitivity is limited by the antibody's affinity (Ka approximately 10(8) M-1) rather than the sensitivity of the electron spin resonance spectrometer (approximately 1 X 10(-7) M). The inhibition of liposome lysis is hapten specific: (epsilon-Dnp-aminocaproic acid,epsilon-Dnp-lysine) greater than alpha-Dnp-glycine; o-nitroaniline and epsilon-dansyl-lysine are ineffective. Inhibition is quantitative without augmentation.

Animals↗
PubMed2001

Systemic complement depletion diminishes perihematomal brain edema in rats.

BACKGROUND AND PURPOSE: The complement cascade is activated after experimental intracerebral hemorrhage (ICH). It remains unclear, however, whether depleting the complement system will improve injury resulting from ICH. This study investigated the effects of systemic complement depletion on brain edema formation after ICH. METHODS: Fifty-six pentobarbital-anesthetized Sprague-Dawley rats were used. Treatment animals were complement-depleted with cobra venom factor (CVF) (intraperitoneally). Control rats received an equal volume of saline injection (intraperitoneally). In both treatment and control rats, autologous blood (100 microL) was infused stereotaxically into the right basal ganglia. Rats were killed 2, 24, or 72 hours later for brain water, ion, and tumor necrosis factor-alpha (TNF-alpha) measurements, for Western blot analysis, and for immunohistochemical studies. Brain edema was quantitated by wet/dry weight. TNF-alpha levels were measured by enzyme-linked immunosorbent assay. Western blot analysis was applied for C9 semiquantification. Immunohistochemistry was used to detect complement C3d, C5a, C9, and myeloperoxidase. RESULTS: Perihematomal brain edema was reduced by systemic complement depletion at 24 hours (78.8+/-0.6% versus 81.5+/-0.8% in control, P:<0.01) and 72 hours (81.5+/-1.5% versus 83.6+/-0.9% in control, P:<0.05), while cerebellar water content was unaffected (78.2+/-0.3% versus 78.0+/-0. 1%). Complement depletion reduced TNF-alpha production 2 hours after ICH. Immunocytochemistry showed that complement depletion significantly reduced perihematomal C9 deposition, C3d production, and the number of C5a- and myeloperoxidase-positive cells. CONCLUSIONS: Complement depletion by CVF attenuates brain edema in ICH, indicating that complement activation plays an important role in ICH-induced brain edema. Preventing complement activation may be effective in the treatment of ICH.

Animals↗
PubMed1994

Effect of thrombosis on complement activation and neutrophil degranulation during in vitro hemodialysis.

Coagulation proteins are known to affect the complement system and neutrophils. To assess the influence of thrombosis on complement and neutrophils during hemodialysis, whole human blood obtained from normal donors and anticoagulated with either 0.8 or 2.0 U/mL of heparin was recirculated for 4 h through cellulose acetate hollow fibers in vitro. Plasma fibrinopeptide A (FPA) levels were measured to assess thrombosis, whereas the activation of the complement system was evaluated at various stages by determining plasma C3a(desArg), C5a(desArg), and SC5b-9 concentrations. The activation of circulating neutrophils was assessed by quantitation of the extracellular release of the intragranular proteins elastase and lactoferrin. Thrombosis was observed when 0.8 U/mL of heparin was used, as indicated by a 36-fold increase in (FPA) levels at 240 min of recirculation and occasional grossly visible blood clots. In contrast, no increase in FPA or clot formation was observed with 2.0 U/mL of heparin. The higher dose of heparin was also associated with a lesser increase in plasma C3a(desArg) and C5a(desArg) concentrations, an observation that is compatible with either an inhibitory effect of heparin or a stimulatory effect of coagulation on the complement system. Plasma elastase or lactoferrin concentrations increased during hemodialysis but were not dependent on heparin dosage at any time. It was concluded that anticoagulation with higher heparin concentration inhibits complement activation in the hemodialysis circuit, but it does not affect neutrophil degranulation.

Anticoagulants↗
PubMed1994

Occurrences, immunoglobulin classes, and biological activities of antibodies in normal human serum that are reactive with Cryptococcus neoformans glucuronoxylomannan.

Serum obtained from normal human subjects contains antibodies reactive in an enzyme-linked immunosorbent assay with the glucuronoxylomannan (GXM) of Cryptococcus neoformans. The frequency of occurrence of class-specific antibodies among normal subjects was 28% for immunoglobulin G (IgG), 98% for IgM, and 3% for IgA. Anti-GXM antibodies with kappa light chains occurred in 98% of normal subjects, while the occurrence of lambda light chains was 28%. Each of five subjects with high levels of anti-GXM IgG antibodies had readily detectable antibodies of the IgG2 isotype; two of the five subjects had readily detectable IgG1 antibody. An examination of sera from human immunodeficiency virus-infected patients showed that human immunodeficiency virus infection was accompanied by a significant decrease in the occurrence of IgM antibodies and anti-GXM antibodies with kappa light chains; these decreases occurred early in infection when CD4 counts were still > or = 500 cells per microliter. A slight but not statistically significant decrease in the occurrence of anti-GXM IgG antibodies was seen only in patients with CD4 levels of < 200 cells per microliter. Sera from normal subjects with high levels of anti-GXM IgG antibodies were examined to identify any contribution of the antibodies to complement activation or to opsonization of the yeast cells. An analysis of the kinetics for activation and binding of C3 to the yeast cell showed no pattern of quantitative or qualitative differences between sera with high or low levels of anti-GXM IgG antibodies. Phagocytosis studies showed that the naturally occurring IgG antibodies did not contribute to opsonization of the yeast cells.

Antibodies, Fungal↗
PubMed2002

Kinetic analysis of glycoprotein C of herpes simplex virus types 1 and 2 binding to heparin, heparan sulfate, and complement component C3b.

Glycoprotein C (gC) from herpes simplex virus (HSV) facilitates virus entry by attaching the virion to host cell-surface heparan sulfate (HS). Although gC from HSV-1 (gC1) and from HSV-2 (gC2) bind to heparin, gC2 is believed to play a less significant role than gC1 in attachment of virus to cells. This attachment step is followed by the binding of gD to one of several cellular receptors. gC also plays an important role in immune evasion by binding to the C3b fragment of the third component of the host complement system. Yet, although both gC1 and gC2 protect HSV against complement-mediated neutralization, only gC on HSV-1-infected cells acts as a receptor for C3b. We used optical biosensor technology to quantitate the affinities (K(D)) and the stabilities (k(off)) between both serotypes of gC with heparin, HS, and C3b to address three questions concerning gC interactions. First, can differences in affinity or stability account for differences between the contributions of HSV-1 and HSV-2 gC in attachment? Our data show that the gC2-HS complex is highly unstable (k(off) = 0.2 s(-1)) compared to the gC1-HS complex (k(off) = 0.003 s(-1)), suggesting why gC2 may not play an important role in attachment of virus to cells as does gC1. Second, does gC2 have a lower affinity for C3b than does gC1, thereby explaining the lack of C3b-receptor activity on HSV-2 infected cells? Surprisingly, gC2 had a 10-fold higher affinity for C3b compared to gC1, so this functional difference in serotypes cannot be accounted for by affinity. Third, do differences in gC-HS and gD-receptor affinities support a model of HSV entry in which the gC-HS interaction is of lower affinity than the gD-receptor interaction? Our biosensor results indicate that gC has a higher affinity for HS than gD does for cellular receptors HveA (HVEM) and HveC (nectin-1).

Binding Sites↗
PubMed1971

Quantitative in vitro measurement of simian virus 40 tumor-specific antigens.

Humoral antibodies, specific for the SV40 tumor-specific transplantation antigen, can be detected by an in vitro cytotoxic assay. (51)Cr-labeled SV40-transformed target cells are selectively killed in the presence of specific antibody and rabbit complement. Inhibition of cytotoxicity can be used to measure the antigen concentration and specific activity on different SV40-transformed cell lines. The assay described has several advantages when compared to other methods currently available to detect the specific SV40 antigen: it is simple, rapid, objective, reproducible, and quantitative.

Animals↗
PubMed1977

Attachment and ingestion of mycoplasmas by mouse macrophages. I. Kinetics of the interaction and effects on phagocyte glucose metabolism.

Rates of attachment and ingestion of Mycoplasma pulmonis by mouse peritoneal macrophages and the effect of the organism on phagocyte glucose metabolism were studied in vitro. Antimycoplasma antibody, but not complement, enhances attachment of mycoplasma to macrophages. Antibody-mediated attachment is not affected by the length of time the cell is maintained in culture or its adherence to a glass surface. Adherence of mycoplasma by nonimmunologic means to cell spread on glass is significantly greater than attachment to macrophages in suspension. Mycoplasmas attached to macrophage membrane do not increase glucose metabolism via the hexose monophosphate shunt. Because of this, the rate of glucose metabolism by macrophages is an accurate quantitative correlate of the recognition and ingestion stage of phagocytosis of mycoplasmas. Antibody induces a more rapid rate of particle ingestion than does altering the mycoplasma by proteolysis. Complement does not augment the rate of ingestion induced by antimycoplasma antibody. By analogy with enzyme and absorption kinetics, it appears likely that differences observed between phagocytosis induced by antibody and that induced by trypsin are due to different rates of recognition between particles rather than different ingestion mechanisms.

Animals↗
PubMed1986

Synthesis of complement components (C3, C2, B and C1-inhibitor) and lysozyme by human monocytes and macrophages.

The synthesis of C3, C2, factor B (B) C1-inhibitor and lysozyme has been studied in monocytes and macrophages isolated from the synovial fluids of patients with rheumatoid arthritis. Concentrations of all 5 proteins in culture supernatants were measured by the sandwich ELISA technique. Kinetic studies showed that only lysozyme and C3 could be detected in monocyte culture supernatants on the first day of culture, whereas C2, B and C1-inhibitor were not present until the third day. In contrast all 5 proteins could be detected in the supernatants of macrophage cultures on day 1. In both monocyte and macrophage cultures synthesis of lysozyme and C1-inhibitor continued throughout the culture period, whereas synthesis of C2, B and C3 appeared to be reduced after the fifth day in culture. Quantitative studies showed that the secretion rates of lysozyme (4,700 X 10(3) molecules/cell/hr) was similar in monocytes and macrophages. Synthesis rates for all 4 complement components in monocyte cultures were less than 0.2% of that for lysozyme. Although the synthetic rates were higher in macrophages, even then they constituted less than 2% of the rate for lysozyme. Synthetic rates for complement components, but not lysozyme, were increased by BSA-anti-BSA antigen-antibody complexes and reduced by serum-treated complexes. Although the functional activity of monocyte B was similar to that for serum, the activity of monocyte C2 was 5 times that of serum C2. As C42 formed with monocyte C2 had a half-life of 13.5 min at 30 degrees C, compared with 4.5 min for the enzyme formed with serum C2, it is probable that monocyte C2 is oxidized by the oxygen products of these cells.

Antigen-Antibody Complex↗
PubMed1982

Analysis of early antigenic changes on heterokaryons between L-cells and a teratocarcinoma-derived cell line, TerC.

A method is presented for the detection of antigenic changes in heterokaryons during the first 24 h after fusion, as detected by antibody- and complement-mediated inhibition of 125IDU uptake. The method employs a variation of the HAT selection protocol, and a mathematical model which allows the reactions of a minority population of heterokaryons to be distinguished from those of the parental cells. This procedure is applied to fusions of clones of the teratocarcinoma-derived cell line TerC, which demonstrate very low levels of H-2b expression, with an L-cell derivative rich in H-2k antigens. Heterokaryons demonstrate an initial tendency for levels of H-2b and H-2k expression to converge, beginning within a few hours of fusion; however, H-2b expression never attains the levels of H-2k, even in long-term hybrids. These results are largely confirmed by quantitative absorptions, except that no diminution of H-2k expression is observed, suggesting that fusion with the TerC parents may transfer a resistance to complement damage to the L-cell parent.

Animals↗
PubMedDate not supplied

Periodic elevation of complement activation products in peritoneal dialysis effluent.

Peripheral white blood cells (WBCs) of chronic ambulatory peritoneal dialysis (CAPD) patients show a reduced expression of chemotactic receptors and a desensitization to chemotactic factor induced events. Chronic complement activation has been suggested as a cause of such phenomena, resulting from either uremia, the indwelling peritoneal catheter, and/or dialysis fluid stimulation. The presence of complement activation products in peritoneal dialysis effluent was assessed by longitudinal measurement of C3bi in eight patients over a 6 to 8 month period, while peritoneal polymorphonuclear leukocyte (PMN) and monocyte CR3 and C5a receptor expression was quantitated by flow cytometry. In six of eight patients, C3bi levels were stable over time and usually measured less than 5 micrograms/ml. Two other patients showed periodic elevation of C3bi, which was not associated temporally with peritonitis. Further study is required to identify the origin of such activated complement components within the peritoneal cavity. However, no correlation was found between dialysate C3bi levels and peritoneal PMN or monocyte expression of CR3 or C5a receptors. Furthermore, the incidence of peritonitis for the two individuals with elevated C3bi was discordant (1 episode/8 patient months vs. 0 episodes/32 patient months), suggesting little or no relationship between these parameters.

Adult↗
PubMed2005

Downregulation of TS, DPD, ERCC1, GST-Pi, EGFR, and HER2 gene expression after neoadjuvant three-modality treatment in patients with esophageal cancer.

BACKGROUND: To find out if neoadjuvant therapy could alter tumor response determinants that might affect tumor sensitivity to the treatment, we investigated intratumoral expressions of genes associated with chemosensitivity, radiosensitivity, or both before and after radiochemotherapy. STUDY DESIGN: Twenty-four patients with locally advanced, resectable esophageal cancer (cT2-4,Nx,M0) received neoadjuvant 5-FU/cisplatin/36 Gy treatment followed by transthoracic en bloc esophagectomy. Expression levels of thymidylate synthase, dihydropyrimidine dehydrogenase, excision repair cross-complementing gene 1 , glutathione S-transferase Pi, epidermal growth factor receptor, and HER2 were measured in matched preradiochemotherapy endoscopic tumor biopsies and in postradiochemotherapy resection specimens. mRNA was isolated from formalin-fixed, paraffin-embedded, laser microdissected tumor tissues, and a quantitative fluorescent dye real-time reverse transcription polymerase chain reaction system was used for gene expression measurement. RESULTS: There was a significant reduction in the expression levels of thymidylate synthase (p < 0.01), dihydropyrimidine dehydrogenase (p = 0.03), excision repair cross-complementing gene 1 (p < 0.01), glutathione S-transferase Pi (p = 0.03), HER2 (p < 0.01), and epidermal growth factor receptor (p = 0.04). The change in the levels of epidermal growth factor receptor mRNA in post- compared with pretreatment specimens was significantly associated with the histopathologic grade of regression (p = 0.01). CONCLUSIONS: The expression levels of a set of genes that are possible determinants of 5-FU/cisplatin/radiation therapy antitumor activity are significantly downregulated by neoadjuvant radiochemotherapy in esophageal cancer.

Adenocarcinoma↗
PubMed1998

Beclomethasone relative availability of oral versus inhaled beclomethasone dipropionate from an HFA-134A metered dose inhaler.

3M has formulated a new chlorofluorocarbon-free (CFC-free) beclomethasone dipropionate (BDP) metered-dose inhaler (MDI) with the use of the propellant HFA-134a (HFA). Lung deposition studies demonstrated that the HFA BDP MDI delivers to the lungs approximately 56% of the BDP dose (ex-adaptor), a substantially higher percentage than the 5-30% delivered by conventional CFC BDP MDIs. As new sensitive bioanalytical methods are becoming available to quantitate systemic levels of inhaled corticosteroids, pharmacokinetic evaluations are emerging as sensitive and reproducible methods that can be used as a complement to the data obtained from lung deposition studies to assess and compare the performance of MDIs. The present study was designed to determine the beclomethasone (BOH) availability of oral BDP relative to inhaled HFA BDP as a first step to alloy MDI product comparisons in the future. Forty mild asthmatic patients completed this open-label, randomized, single-dose, two-period crossover study. Each patient received an oral dose of BDP (0.2, 0.5, 1, 2 or 5 mg) in one period and an inhaled dose of BDP (0.2 or 0.8 mg) in the other period, with four patients allocated to each of ten different treatment sequences. The BOH availability of orally administered BDP was approximately 40% relative to inhaled HFA BDP. In addition, the fraction of an oral dose that reaches the systemic circulation was estimated from the 40% relative availability and previous lung deposition data to be 0.26. These estimated pharmacokinetic parameters will be used in the future to further characterize the pharmacokinetics of inhaled BDP and to compare the performance of different MDI products.

Administration, Inhalation↗
PubMed1980

Major histocompatibility gene complex (MHC)-coded determinants on antigen-specific suppressor factor for delayed-type hypersensitivity and surface phenotypes of cells producing the factor.

Antigen-specific suppressor factor for delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) was obtained by incubating in vitro spleen cells from CBA mice (H-2k) injected intravenously 3 days previously with 1 x 10(9) SRBC. The suppressor factor was characterized for major histocompatibility gene complex (MHC)-coded antigenic determinants by passing the factor through immunosorbents coupled with appropriate alloantisera. The suppressor factor was absorbed by anti-H-2k, anti-Iak and anti-I-Jk immunosorbents but was not retained by anti-Ias, anti-I-Js, anti-I-Ak, anti-I-E/Ck or anti-H-2Kk immunosorbents. In addition, the factor bound to an immunosorbent coupled with rabbit antibodies against carbohydrate-defined Ia antigens. Furthermore, the suppressive activity that was absorbed was quantitatively recovered in the acid eluates from the immunosorbents. Treatment of the spleen cells with anti-Lyt-1.1 antiserum and complement completely abrogated their ability to elaborate the suppressor factor in vitro. In contrast, treatment with anti-Lyt-2.1 or anti-Iak antiserum and complement had no effect. Thus, it appears that the suppressor factor for DTH to SRBC bears I-J subregion-coded determinants, and its production is dependent on cells which have the Lyt-1+,2- and Ia- phenotype.

Animals↗
PubMed1998

Abnormal rearrangements associated with V(D)J recombination in Fanconi anemia.

The hallmark of Fanconi anemia (FA), a rare inherited cancer prone disorder, is a high level of chromosome breakage, spontaneous and induced by cross-linking agents. The increased genomic instability of FA is reflected at the gene level by an overproduction of intragenic deletions. Two of the eight FA genes have been cloned, however, their function remains unknown. We recently demonstrated that the lack of functional FA genes lead to a marked decrease in the fidelity of non-homologous end-joining, a pathway that mammalian cells predominantly use to repair DNA double-strand breaks (DSB). Knowing that specific DSB are generated during V(D)J recombination, here we have examined the molecular features of V(D)J rearrangements in normal and FA lymphoblasts belonging to complementation groups C and D. Using appropriate extrachromosomal recombination substrates, V(D)J coding and signal joint formation have been analysed quantitatively and qualitatively. Our results show that the frequency of coding and signal joint formation was not significantly different in normal and FA cells. However, when the fidelity of the V(D)J reaction was examined, we found that in normal human lymphoblasts V(D)J recombination proceeds with high precision, whereas, in FA cells a several fold increase in the frequency of aberrant rearrangements is associated with V(D)J coding joint formation. The abnormal recombinants that we recovered in FA are consistent with excessive degradation of DNA ends generated during the V(D)J reaction. On the basis of these findings, we propose a working model in which FA genes play a role in the control of the fidelity of rejoining of specific DNA ends. Such a defect may explain several basic features of FA, such as chromosomal instability and deletion proneness.

Cell Line↗
PubMed1993

Algebraic analysis of the determinants of VO2,max.

Considerable evidence exists to support the idea that maximum oxygen consumption in exercising mammals is determined by the quantitative interaction between diffusive and convective factors involved in the transport of oxygen between the environment and the muscle mitochondria. To complement experimental tests of this hypothesis as well as numerical modeling of the interaction between diffusion and convection, both in the lungs and tissues, this paper reports an analytical (algebraic) model of diffusive/convective interaction made possible by replacing oxygen by a hypothetical gas of similar effective solubility, obeying Henry's law. The model describes a homogeneous but potentially diffusion-limited lung arranged in series via the circulation with a homogeneous but potentially diffusion-limited muscle. Steady-state transport of gas through this system and its subsequent tissue utilization are described by three equations: (1) mass balance across the lungs, (2) diffusive transport across the lungs, and (3) diffusive transport in the tissues. The latter two equations involve interaction between diffusive and perfusive (convective) elements of gas transport. Independent variables in this analysis are inspired partial pressure, alveolar ventilation, cardiac output, effective solubility of the gas, and diffusing capacities of the lungs and of the tissues. Dependent variables are alveolar, arterial, and venous partial pressures and hence maximum O2 uptake by the tissues (VO2,max). Evaluation of this model in both normoxia and severe hypoxia is described. In normoxia, VO2,max is shown to be affected by all independent variables, but mostly by blood flow. However, in severe hypoxia, VO2,max, based on data from Operation Everest II, becomes more sensitive to muscle diffusing capacity and less so to blood flow. In normoxia, normal sea level values of the independent variables set VO2,max at a point where little further gain in VO2 would be seen without large increases in diffusive or convective properties. On the other hand, there is relatively little reserve, such that decreases in transport parameters would result in significant reductions in VO2,max.

Animals↗
PubMed1975

Segmental ultrasonic test for the study of cholecystodyskinesia.

Ten patients of ages 35 to 61 years with 'cholecystopathy' received an injection of 20 ml, 40% biligrafin. X-rays of the gall-bladder area were taken before and at certain intervals after the injection. This was followed by ultrasonic stimulation (applied using the impulse technique) for ten to twenty minutes at frequencies of 3 MHz, effective intensity of 1.4-2.8 W cm-2 and with paraffin oil as a contact agent. The dorsal segments were subjected to ultrasonic stimulation of weaker intensity but longer duration than the ventral segment. The gall-bladder shadow was analysed planimetrically, and an approximate volumetric calculation was attempted. The results show that the gall-bladder responds to segmental ultrasonic stimulation by both contraction and dilatation, presumably through a cutaneous visceral reflex arch, a qualitatively and quantitatively different response to that of egg yolk. Segmental ultrasonic stimulation of the gall-bladder seems to be a suitable complement to fatty meal treatment, particularly where this 'contra-indicated'. In addition it offers the possibility of differentiating between hyper and hypokinetic forms of gall-bladder dyskinesia. Finally, it was observed that a more marked effect on the gall-bladder can be produced by dorsal-segmental stimulation than by stimulation over the ventral points.

Adult↗
PubMed1996

The relative abundance of dopamine D4 receptor mRNA in post mortem brains of schizophrenics and controls.

An increase in dopamine D4 receptors has recently been reported in post mortem brain samples from schizophrenics. We have attempted to complement this finding by assessing the levels of the specific messenger RNA (mRNA) for the D4 receptor, using the technique of quantitative RNA-PCR. No significant differences in the levels of expression of mRNA for the D4 receptor were found in the brains from schizophrenics compared to controls. The relationship between D4 receptors and schizophrenia, therefore, remains unclear.

Adult↗
PubMed2005

Enzymology of the anaphase-promoting complex.

The anaphase-promoting complex (APC) is an ubiquitin-protein ligase that promotes mitotic progression by catalyzing the ubiquitination of numerous proteins, including securin and cyclin. Its complex subunit composition and extensive regulation make the APC an active subject of investigation for both cell biologists and enzymologists. This chapter describes a system for the reconstitution and quantitative analysis of APC activity from budding yeast in vitro. We focus in particular on the measurement of processive ubiquitination, which complements traditional analysis of the reaction rate as a means to elucidate the molecular details of substrate recognition and ubiquitination by the APC.

Anaphase-Promoting Complex-Cyclosome↗