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PubMed1989

Staphylococcal protein A adsorption in neoplastic disease: analysis of physicochemical aspects.

Staphylococcus aureus organisms and immobilized Staphylococcal protein A have been used as extracorporeal immunoadsorbents for the treatment of neoplastic disease. In a significant number of cases, a biologic response has been documented. This review analyzes the experimental results to date, correlates experimental design parameters with response to treatment, and discusses possible mechanisms of action. The evidence implicates protein A as the tumoricidal agent, although bacterial or complement components may act independently or synergistically. A more quantitative and fundamental approach is needed to exploit the potential for protein A immunoadsorption in treating neoplastic disease.

Adsorption↗
PubMed1985

Transfer of tubulointerstitial nephritis in the Brown Norway rat with anti-tubular basement membrane antibody: quantitation and kinetics of binding and effect of decomplementation.

Lewis (LEW) rats immunized with Brown Norway (BN) rat renal basement membrane (RBM) and adjuvants produce high titer circulating anti-BN tubular basement membrane (TBM) antibodies, in addition to developing an autoimmune cell-mediated form of nodular tubulointerstitial nephritis (TIN). This immune LEW serum, which reacted with BN TBM but not LEW TBM by immunofluorescence, was capable of passively transferring TIN as early as 24 hr after administration of volumes as low as 3 ml i.v. to normal BN recipients, producing focal lesions histologically and immunopathologically similar but less extensive than those studied previously in this strain after active immunization with heterologous RBM. In contrast, a total of 45 ml of serum (in multiple doses) from BN rats immunized with bovine RBM and adjuvants produced only one small lesion of TIN in a recipient BN rat. This difference in serum transferability of anti-TBM-associated TIN appears to relate to quantitative differences in anti-particulate and soluble (collagenase-extracted) BN RBM antigen reactivity measured by radioimmunoassay. Paired-label quantitative studies of passively transferred LEW anti-BN RBM IgG demonstrated a slow accumulation of renal-bound antibody over 6 days, and corresponded with kidney elution and immunofluorescence studies after transfer of immune LEW sera to normal BN rats. Approximately 167 micrograms of kidney-fixing antibody per gram of kidney were calculated to be required for the development of the earliest cellular infiltration. C3 depletion with cobra venom factor greatly diminished the development of destructive TIN lesions associated with multinucleate giant cells after passive transfer of LEW anti-BN RBM antibody to BN rats. This study, using immune LEW sera containing high levels of anti-BN RBM antibody, has defined and quantitated a role for anti-TBM antibody and complement in the initiation of TIN in BN rats.

Animals↗
PubMed1984

Effect of pregnancy on augmentation of natural killer cell activity by Corynebacterium parvum and Toxoplasma gondii.

Studies were performed to determine the effect of pregnancy on NK cell activity and the augmentation of NK cell activity which normally follows injection of killed C. parvum or live T. gondii. When compared to cells from virgin mice, peritoneal and splenic cells from unstimulated pregnant mice showed no significant difference in NK activity. In contrast, NK activity of peritoneal cells from pregnant mice that received C. parvum or T. gondii was significantly lower than that found in comparably treated virgin mice. This reduced augmentation of NK activity in the peritoneal cells of pregnant mice could not be altered by the removal of plastic-adherent cells or of T cells by treatment with anti-Thy-1.2 antibody and complement. The number of effector-target conjugates was quantitated with a single cell assay, and was found to be comparable between cells from treated pregnant mice and treated virgin controls. However, the magnitude of lysis of target cells by peritoneal and splenic effector cells from pregnant mice previously injected with C. parvum was markedly and significantly reduced when compared to cells from comparably treated virgin mice. Thus, pregnancy is associated with an impaired ability to augment NK activity. This decrease in NK activity was not due to the depletion or dilution of NK cells, but to a functional impairment of the individual NK cells.

Animals↗
PubMed1997

[Cerebral infarct and the immune response].

There are only few data available regarding the immunological mechanisms for cerebral infarction. The aim of this study was to find out the humoral and cell-mediated immunity under the conditions of focal brain ischemia (CI). As a method for humoral immunity, the complement consumption test against a panel of 8 antigens, quantitative analysis of immunoglobins and fractionized sedimentation of erythrocytes were used in the group of pts with CI, and the group of atherosclerotics (AS) and hypertonics (VH), potential victims of focal brain ischemia. It was found that the occurrence of antibodies against the whole panel of antigens in the group of CI is significantly higher as compared with the healthy controls, but it is lower than that in the group of AS and VH. The occurrence of antibodies exclusively against only brain antigens and that in CSF is similar. No correlation to the location of ischemic lesion and the degree of neurological deficit score was found. These findings didn't change in 2 and 4 weeks as well as in 1 year after the onset of CI. The quantitative analysis of immunoglobins revealed statistically higher levels of IgA and lower levels of IgM in comparison with the controls. IgG were higher, but without statistical significance. Statistically significant higher levels of all immunoglobins in CSF were found. As similar trend of changes found also in the group of AS and VH. These results of humoral immunity confirmed by the results of fractionized sedimentation of erythrocytes with EP. The results can be interpreted as a possible change or disorder of central regulation of immunizing processes due to the latent (in AS and VH) of manifest (in CI) lesions of the brain. But the quality and quantity of this response might have been affected by the entire case history of the patients who survived cerebral infarction. The changes in immunity response of the organism in CI was shown also in cell-mediated immunity. The results a statistically significant increase in stimulatory (SI) as well as in immunoregulatory (IRI) indices in stroke patients under the age of 40. These findings didn't change 2 and 4 weeks after the onset of CL. An increase in IRI was due to the increase in Th lymphocytes. In the immune response of the organism in CI, the antiphospholipid antibodies (aPLs = anticardiolipin antibodies (aCL) and lupus anticoagulant--LA) play an important role. aCLs were present in 9.8% of the first stroke pts when compared to 4.3% in controls. The most common isotype of the antibodies we IgG. Of all first-stroke pts who were aCL positive only 8% had no other stroke risk factors (atrial fibrillation, diabetes, hypertension and other). aCLs are an important risk factor for the first stroke, mainly in the young, but also in the elderly. The presence of aCLs increases the risk for recurrent strokes. aPLs are not necessarily associated with the specific location of clinical stroke syndrome but they are in significant correlation to the occurrence of multiple strokes on CT (30:18%). None of the initially aCL-negative patients became aCL-positive during the time course of CI. These data support the idea that aCLs play a causal role in stroke (PROPTER HOC changes) rather than vice versa (POST HOC changes). From the therapeutic point of view, currently there do not exist any good treatment guidelines for preventing the second stroke. The analysis of HLA. antigen showed an increase in some HLA (A2, A28 etc.) and a decrease in others (A3, A9 etc.) in comparison with the controls. This might refer to the participation of genetic factors in the onset of CI.

Adult↗
PubMed2004

BF, HP, DQB and DRB are associated with haemolytic complement activity, acute phase protein reaction and antibody response in the pig.

In order to examine the loci factor B (BF), C3, corticotropin releasing hormone (CRH), DQB, DRB, haptoglobin (HP) and transforming growth factor beta 1 (TGFB1) for association with traits of humoral, specific and unspecific defence F2-animals of a porcine resource family were genotyped at single nucleotide polymorphisms within these loci. Haemolytic complement activity in the alternative and classical pathway, C3c and haptoglobin serum concentration and antibody titres were determined immediately prior and at days 4 and 10 after vaccinations against Mycoplasma hyopneumoniae (Mh), Aujeszky's disease virus, and porcine reproductive and respiratory syndrome virus at 6, 14 and 16 weeks of age, respectively. Analysis of variance revealed association of BF, HP and DRB with C3c serum concentration. The trend of haemolytic complement activity and C3c serum concentration during the experiment was affected by the interaction of DQB genotype and time of measurement. Association with antibody titres were found for BF, DQB and DRB. Results of the mixed model analyses were confirmed by quantitative transmission disequilibrium test that showed linkage and association with antibody titres, complement activity and acute phase reaction at certain times of measurement. The findings promote the importance of the candidate genes for humoral mechanisms of unspecific and specific defence that provide natural resistance against many pathogens.

Acute-Phase Proteins↗
PubMed1987

Activation of the classical and alternative pathways of complement by Treponema pallidum subsp. pallidum and Treponema vincentii.

Both in vivo and in vitro studies have indicated that complement plays an important role in the syphilitic immune responses. Few quantitative data are available concerning activation of the classical pathway by Treponema pallidum subsp. pallidum, and no information is available on treponemal activation of the alternative pathway. Activation of both pathways was compared by using T. pallidum subsp. pallidum and the nonpathogen T. vincentii. With rabbit and human sources of complement, both organisms rapidly activated the classical pathway, as shown by hemolysis of sensitized sheep erythrocytes and by the generation of soluble C4a. With human sources of complement, both organisms also activated the alternative pathway, as shown by hemolysis of rabbit erythrocytes and by the generation of soluble C3a in the presence of magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). During incubation, organisms remained actively mobile and did not lyse, indicating that activation was a function of complement reactivity with the intact outer treponemal surface. In addition, freshly harvested T. pallidum subsp. pallidum immediately activated both pathways of complement; preincubation of organisms did not enhance complement reactivity. T. vincentii was a more potent activator of this pathway. T. pallidum subsp. pallidum contained almost four times as much surface sialic acid as T. vincentii did. When sialic acid was enzymatically removed from T. pallidum subsp. pallidum, enhanced activation of the alternative pathway was detected. It is proposed that T. pallidum subsp. pallidum retards complement-mediated damage by the alternative pathway through surface-associated sialic acid. This may be an important virulence determinant that enables these organisms to readily disseminate through the bloodstream to infect other tissues.

Animals↗
PubMed1988

Prenatal detection of 46,XY,rec(5),dup q, inv(5)(p13q33) using DNA analysis, flow cytometry, and in situ hybridization to supplement classical cytogenetic analysis.

Distinguishing between balanced and unbalanced chromosome complements segregating from parental rearrangements may be difficult using only classical cytogenetic techniques if banding morphology is similar under both expectations. In these situations, supplementing cytogenetic analysis with molecular genetic techniques and flow cytometry may provide increased diagnostic accuracy. To illustrate this, we present a case in which similar band pattern morphology would be expected for both the balanced carrier (heterozygote) and the recombinant dup q chromosome complements segregating from a mother with a balanced inversion [46,XX,inv(5)(p13q33)]. The parents came to Northwestern for consultation after receiving conflicting interpretations of their first amniotic fluid cultures. An ultrasound examination was said to be normal. They inquired whether there were ways to increase their confidence that the complement was unbalanced. Their reluctance to terminate the pregnancy was due to a 6-year history of infertility. After extensive counselling, the couple elected repeat amniocentesis. Further cytogenetic analysis of repeat amniotic fluid cultures by G-banding and R-banding, molecular genetic analysis with highly polymorphic DNA probes, and quantitative flow cytometry were performed. Results agreed that an unbalanced fetal complement was present. Southern blot analysis with a 5p marker definitively demonstrated a lack of maternal 5p material in the fetus, and in situ hybridization showed a 5q marker at either end of the recombinant chromosome. Flow cytometry was consistent with this interpretation. Because of the advanced gestational age, the parents elected to terminate based on cytogenic results of the second amniocentesis, rather than to wait another 1-2 weeks for results of other methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2004

The genetic architecture of quantitative traits: lessons from Drosophila.

Understanding the genetic architecture of quantitative traits begins with identifying the genes regulating these traits, mapping the subset of genetically varying quantitative trait loci (QTLs) in natural populations, and pinpointing the molecular polymorphisms defining QTL alleles. Studies in Drosophila have revealed large numbers of pleiotropic genes that interact epistatically to regulate quantitative traits, and large numbers of QTLs with sex-, environment- and genotype-specific effects. Multiple molecular polymorphisms in regulatory regions of candidate genes are often associated with variation for complex traits. These observations offer valuable lessons for understanding the genetic basis of variation for complex traits in other organisms, including humans.

Animals↗
PubMed1967

Serological comparison of spherules and arthrospores of Coccidioides immitis.

Spherule and arthrospore cellular preparations were sonic-treated and separated into their respective supernatant and sediment components. Complement-fixation tests with antispherule and antiarthrospore pooled rabbit sera revealed that the soluble antigens exhibited more serological activity than the sediment preparations. After autoclaving, an arthrospore cellular antigen exhibited increased activity with either antisera, whereas autoclaved spherules exhibited increased activity only with antispherule serum. Complement-fixation tests with coccicioidin and spherule culture supernatant preparations revealed quantitative or qualitative differences in antigenic determinants between these two morphological phases of Coccidioides immitis.

Antigen-Antibody Reactions↗
PubMed1980

Digital integration of granulocyte aggregation responses. A simple and reproducible method for the quantitation of granulocyte adhesiveness.

The pulmonary leukostasis and lung dysfunction associated with intravascular complement activation results from C5a-mediated granulocyte (GR) aggregation, a phenomenon which can be reproduced in vitro using standard nephelometric techniques. To produce a more subjective measure of the extent and rate of GR aggregation responses we added a digital integrator to the system. The validity of this approach was substantiated by the close correlation between the aggregating and chemotactic activities of C5a and N-formyl-methionine-leucine-phenylalanine. Use of this technique enabled us to define the dose-response relationship of the aggregation produced by the cationophore A23187 and the inhibitory effect of tetracaine on divalent cation-dependent aggregation responses. The aggregation produced by these three stimuli does not result primarily from simple cross-linking of surface changes because, unlike the passive cell-cell association produced by the cation poly-L-lysine, it is not inhibited by anionic poly-L-glutamic acid. The importance of microtubules as regulators of GR adhesiveness was substantiated by the inhibitory effects of colchicine (but not lumicolchicine) on aggregation in this system. These data suggest that this integration of light transmission increments is a useful adjunct to this basic technique, whether used as a bioassay for chemotactic stimuli or as a model to study the many factors which regulate GR adhesiveness.

Calcimycin↗
PubMed1985

Platelet associated complement components (PAC3c and PAC3d) in patients with autoimmune thrombocytopenia.

Platelet associated C3c and C3d (PAC3c and PAC3d) were quantitated by enzyme linked assay in 105 patients with idiopathic autoimmune thrombocytopenia (AITP) in whom elevated platelet associated immunoglobulins (IgG and IgM) had previously been documented. Increased levels of complement components were demonstrated in 46 of 105 patients (43.8%). In 11 of these patients, PAC3d alone was abnormal implying that C3b had been inactivated after cleavage by C3 inactivator in vivo. Complement binding was seen in two of 16 patients (12.5%) with raised PAIgG alone, four of 19 patients (21.0%) with raised PAIgM alone and in 40 of 70 patients (57.1%) in whom PAIgG and PAIgM were raised together. This difference was highly significant (P = 0.01). The clinical implication of these findings are discussed.

Autoimmune Diseases↗
PubMed2003

Rat glomerular epithelial cells produce and bear factor H on their surface that is up-regulated under complement attack.

BACKGROUND: Factor H is a potent complement inhibitory molecule that is primarily produced by the liver and appears in plasma as a soluble protein. Yet there is evidence that other cells, including those in the kidney, can produce factor H, and that it can be cell-associated as well as present as a plasma protein. Here we studied factor H in rat glomerular epithelial cells (GEC). METHODS: A polyclonal antibody to factor H was used to identify factor H protein. A polymerase chain reaction (PCR)-based strategy was utilized to clone the full-length cDNA of GEC factor H. The relative quantity of factor H mRNA was measured by quantitative reverse transcription (RT)-PCR in cultured GEC exposed to complement activation and in the passive Heymann nephritis (PHN) model of membranous nephropathy. RESULTS: By immunofluorescence microscopy, factor H protein was present on the plasma membranes of cultured GEC. Based upon Western blot studies, this appeared to be the full-length 150 kD factor H protein. Factor H cDNA cloned from GEC was identical to the newly deposited sequence for rat liver factor H cDNA. In cultured GEC in which complement was activated, factor H mRNA increased over time. Similarly, in the PHN model in which complement was activated on GEC in vivo, factor H mRNA and protein also increased over time. CONCLUSION: Cultured GEC and glomeruli express factor H mRNA and protein. As modeled both in vitro and in vivo in the rat, factor H is up-regulated in membranous nephropathy. This is likely to be a direct response of GEC to complement attack and may represent a protective response of this cell.

Animals↗
PubMed1986

SDS denaturation of complement factor C3 as a model for allosteric modifications occurring during C3b binding: demonstration of a profound conformational change by means of circular dichroism and quantitative immunoprecipitation.

The antigenic expression of bound but not fluid-phase C3b closely resembles that of sodium dodecyl sulphate (SDS) denatured C3. For this reason, denatured C3 has been used in this study as a model to characterize the conformational changes associated with bound C3b. It was shown in circular dichroism in the far UV spectrum that profound changes in the secondary structure occurred in denatured C3. Furthermore, quantitation by immunoprecipitation of the previously observed antigenic changes during denaturation demonstrated that C3 lost 2/3 of the antigens associated with native C3 whereas 1/3 were stable. The lost antigens were completely replaced by antigens that are specific for denatured and bound C3. We postulate that the binding of C3b is accompanied by a profound conformational change distinctive of that observed in fluid-phase C3b.

Allosteric Site↗
PubMed2001

Expression of the adhesion molecules Mac-1 and L-selectin on neutrophils in acute pancreatitis is protease- and complement-dependent.

OBJECTIVE: To examine the effect of pancreatic proteases on the expression of the adhesion molecules Mac-1 and l-selectin on neutrophils, and the role of complement activation in this process. SUMMARY BACKGROUND DATA: Sequestration of neutrophils in the pancreatic and pulmonary microvasculature characterizes acute pancreatitis. METHODS: Serum was collected from inbred rats after induction of necrotizing pancreatitis; trypsinogen activation peptide was measured to quantify trypsin activation. Normal rat serum was also collected and subjected to limited trypsin digestion with and without the addition of complement inhibitor. Both groups of sera were incubated in vitro with healthy leukocytes. Expression of Mac-1 and L-selectin on neutrophils was measured quantitatively by flow cytometry. To assess the consequences of these events in vivo, trypsinated serum with or without complement inhibition or control serum was infused intravenous into rats. Soybean trypsin inhibitor was added to serum before injections to block residual trypsin activity. Pancreatic and pulmonary injury was quantitated by histology, measurement of edema, and myeloperoxidase activity. RESULTS: Mac-1 expression on neutrophils incubated with pancreatitis serum was increased compared with controls, whereas L-selectin was decreased. Neutrophils incubated with trypsinated serum also showed upregulation of Mac-1 and downregulation of L-selectin, particularly with trypsin at 10(-4) mol/L. Addition of soluble complement receptor 1 abrogated both Mac-1 upregulation and L-selectin downregulation. Lungs of animals injected with trypsinated serum showed increased edema and myeloperoxidase activity, which were reduced by soluble complement receptor 1. CONCLUSIONS: Trypsin-generated complement activation participates in the upregulation of Mac-1 and shedding of L-selectin on neutrophils in acute pancreatitis. Protease or complement inhibition may be effective in preventing leukocyte migration and subsequent local and remote organ injury.

Analysis of Variance↗
PubMed1973

Nonimmune chemotaxis in vivo: inhibition by complement depletion with cobra factor.

Wistar rats were depleted of complement components using purified cobra venom factor (CoF) administered over a 30-hour period by intraperitoneal injection. Complement depletion was confirmed by immunodiffusion assay, hemolytic assay and inhibition of the reverse Arthus reaction. Rats depleted to below 3% of their normal serum complement level showed marked inhibition of chemotaxis into inflammatory fluid produced in polyvinyl sponges 24 hours after implantation into the dorsal subcutaneous region. Subsequent studies were done to test the effect of high local concentrations of CoF on the microcirculation, the neutrophil and the connective tissue. Local exposure of these tissues and cells to CoF in vivo had no inhibiting effect on chemotaxis. Cobra venom factor did not affect neutrophil survival in vitro or in vivo. The most likely hypothesis is that CoF works by depleting complement and that in vivo chemotaxis of the nonbacterial, nonimmune type is complement dependent to the extent of 60 to 80%. The system described allows for quantitative determination of chemotaxis in vivo.

Animals↗
PubMed2005

Effect of glycosylphosphatidylinositol specific phospholipase D gene expression levels on complement mediated killing of leukemic cells in patients with chronic myeloid leukemia.

BACKGROUND: To explore the disparity in glycosylphosphatidylinositol phospholipase D (GPI-PLD) expression levels between mononuclear cells of chronic myeloid leukemia (CML) and healthy controls, and clarify the certain relation of GPI-PLD expression levels to complement mediated killing of leukemic cells. METHODS: Competitive RT-PCR was used to detect quantitatively the GPI-PLD mRNA in mononuclear cells. GPI-anchored CD55 and CD59 were analyzed by flow cytometry and Western blotting. Complement-mediated lysis was assessed by staining method of trypan blue dye. RESULTS: The GPI-PLD activities and their mRNA copies in CML patients were significantly lower than those in healthy adults. At the tenth day after treatment with bone marrow transplantation (BMT), the GPI-PLD activities and copies of GPI-PLD mRNA almost recovered to the expression levels of healthy subjects. The expression of both CD55 and CD59 in CML patients were significantly higher than those in healthy subjects. After treatment with insulin (10(-7) mol/l) plus glucose (16.7 mmol/l) for 48 h, the cellular GPI-PLD activity and mRNA levels in K562 cells derived from the leukemic cells of a CML patient all increased about 3-fold. Simultaneously, the GPI-anchored CD55 and CD59 on cell surfaces were released into the culturing medium, and the killing rate of complement-mediated K562 cell lysis increased almost 3 times. CONCLUSION: The decreased GPI-PLD expression may reduce the release of GPI-anchored CD55 and CD59 in leukemia cells and finally decrease complement mediated killing of these cells in chronic phase of CML.

Adult↗
PubMed2005

Concentration and particle size of airborne toxic algae (brevetoxin) derived from ocean red tide events.

Red tides in the Gulf of Mexico are formed by blooms of the dinoflagellate Karenia brevis, which produces brevetoxins (PbTx). Brevetoxins can be transferred from water to air in the wind-powered whitecapped waves during red tide episodes. Inhalation exposure to marine aerosol containing PbTx causes respiratory problems. A liquid chromatograph/ tandem mass spectrometric method was developed for the detection and quantitation of several PbTxs in ambient samples collected during red tide events. This method was complemented by a previously developed antibody assay that analyzes the entire class of PbTx compounds. The method showed good linearity, accuracy, and reproducibility, allowing quantitation of PbTx compounds in the 10 pg/m3 range. Air concentrations of PbTxs and brevenal for individual samples ranged from 0.01 to 80 ng/m3. The particle size showed a single mode with a mass median diameter between 6 and 10 microm, which was consistent for all of the PbTx species that were measured. Our results imply that individual PbTxs were from the same marine aerosol or from marine aerosol that was produced from the same process. The particle size indicated the likelihood of high deposition efficiency in the respiratory tract with the majority of aerosol deposited in the upper airways and small but not insignificant deposition in the lower airways.

Aerosols↗
PubMed1985

The determination of the complement components C1q, C4 and C3 in serum and cerebrospinal fluid by radioimmunoassay.

Non-competitive 2-site radioimmunoassays (RIA) for the determination of the complement proteins C1q, C4 and C3 in cerebrospinal fluid (CSF) are described. The quantitative results of the RIAs were the same as those obtained by other assay methods: radial immunodiffusion and turbidimetry and, in the case of C4, the haemolytic assay. The concentrations of the complement proteins in paired CSF and serum samples from a group of 60 patients were measured, as well as those of albumin and IgG. The ratios (concentration in CSF)/(concentration in serum) of the complement proteins correlated poorly with that of albumin. In contrast, the ratio of IgG was significantly correlated with that of albumin. The ratios of the complement proteins were higher than might be expected on the basis of their molecular masses. This suggests that these proteins may be synthesized within the normal central nervous system.

Adolescent↗