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PubMed1984

Complement activation during hemodialysis: clinical observations, proposed mechanisms, and theoretical implications.

Human C3a radioimmunoassay techniques were employed to define both the temporal profile and the amount of complement activation taking place in the extracorporeal circuit during maintenance hemodialysis. Prospective studies demonstrated that C3a formation, like hemodialysis-associated leukopenia, was a transient phenomenon that occurred predominantly during the first 30 min of dialysis. Quantitative comparisons revealed that new Cuprophan hemodialyzers displayed somewhat greater complement-activating potential than cellulose acetate dialyzers. By contrast to new Cuprophan membranes, both reused Cuprophan and polyacrylonitrile dialyzers exhibited only a modest ability to activate human complement. These findings are compatible with the known mechanisms of complement activation and suggest that certain chemical and biochemical methods might be exploited to enhance the biocompatibility of cellulose dialysis membranes.

Acrylic Resins↗
PubMed1983

Kinetics of acute inflammation induced by Escherichia coli in rabbits. II. The effect of hyperimmunization, complement depletion, and depletion of leukocytes.

The inflammatory response to Escherichia coli was quantitated in the skin of normal rabbits and the kinetics established as described previously. Hyperemia, measured with radiolabeled microspheres; vascular permeability, estimated with 125 I-albumin; and leukocyte infiltration, quantitated with 51Cr-labeled autologous leukocytes, reached maximal values 3 hours after the injection of bacteria and subsided almost completely by 6 hours. Hemorrhage, measured with homologous 59Fe-erythrocytes, continued to increase between 1 and 6 hours after injection and then reached plateau levels. The lesions were studied up to 8 hours, since in the previous study no changes were observed beyond that time. In the study described in this paper, the host mediation systems were manipulated in various groups of rabbits in order to elucidate the mechanisms underlying the development of the inflammatory reaction. One group of animals was hyperimmunized with the E coli organisms, another was partially depleted of hemolytic complement with cobra venom factor, and yet another was rendered leukopenic with nitrogen mustard. In hyperimmunized animals hyperemia in the dermal lesions induced by the microorganisms was significantly more intense than in normal rabbits. Vascular permeability increase occurred earlier in hyperimmunized rabbits and at 1 hour was significantly greater than in normals. Decomplemented rabbits had significantly less vascular permeability than normal animals, whereas in leukopenic rabbits no increase in vascular permeability could be elicited. Leukocyte accumulation was increased over the normal animals in the lesions of hyperimmunized rabbits. Hemorrhage was significantly decreased in leukopenic rabbits. Histologic examination of the lesions revealed that whereas in normal animals the infiltrating neutrophils ingested most of the bacteria and formed definite abscesses by 6-8 hours, these abscesses were absent in leukopenic animals, and free-lying bacteria were demonstrable in lesions. Histologically more neutrophils were present in the hyperimmunized than in the normal rabbits, but this difference was striking when normal animals were compared with leukopenic animals, in some of which only very occasional small accumulations of neutrophils were present.

Acute Disease↗
PubMed1987

Complement activation in extracorporeal circuits.

Clinical studies performed with various types of hemodialyzers provide a data base and conceptual framework for understanding blood-material interactions that result in complement activation. Current findings suggest that the complement-activating potential of biomaterials may be defined by quantitating either fluid-phase C3a antigen or measurement of surface-bound C3 fragments. Furthermore, systematic studies can reveal material properties that regulate both the production and distribution of the human anaphylatoxins. Assessing the fate of the C5a anaphylatoxin is particularly important because it is likely that generation of this bioactive molecule in extracorporeal circuits initially triggers granulocyte responses and may produce cardiopulmonary manifestations if the C5a is formed in sufficient quantities. It seems likely that detailed knowledge of these varied phenomena will permit rational design of newer "biocompatible" materials and membranes.

Biocompatible Materials↗
PubMed1981

Normal neutrophil function in human renal allograft recipients receiving Minnesota anti-lymphoblast globulin (MAG).

Equine anti-human lymphoblast globulin (MAG) used in combination with other immunosuppressive agents did not impair chemotaxis, phagocytosis, nitroblue tetrazolium (NBT) reduction or halogenation by peripheral blood neutrophils (PMN) from seven human recipients of cadaver renal allografts. MAG was given to the allograft recipients following transplantation, and the dosage was determined each day by quantitation of peripheral blood T and B lymphocytes. No deposition of immunoglobulin or complement on the recipient's PMN was detected by indirect immunofluorescence. Chemotaxis of PMN was determined in blind-well chambers, and phagocytosis of opsonized zymosan was quantitated by spectrophotometric measurement of solubilized formazan, and halogenation was measured by quantitating the incorporation of 125I into PMN that had phagocytosed opsonized zymosan. Chemotaxis, phagocytosis, NBT reduction, and halogenation by neutrophils from patients receiving MAG, prednisone and azathioprine were not impaired.

Adult↗
PubMed1982

Measurement of the complement C3 breakdown product C3d by rocket immunoelectrophoresis.

A method is described for quantitative measurement of C3d in plasma and synovial fluid by the use of rocket immunoelectrophoresis after fractionation of the samples with 22% polyethylene glycol. This method has the advantage over radial immunodiffusion of being more sensitive (detecting C3d down to 3 mg/l) whilst proving equally reproducible. Investigations indicate that the collection of blood samples in EDTA prevents in vitro activation of C3 even after storage for up to 6 h at room temperature and up to 12 weeks at -70 degrees C. Elevated levels of C3d were found in a proportion of SLE and RA plasma samples and in synovial fluids from patients with inflammatory synovitis. It is suggested that C3 conversion in vivo may be assessed by measurement of C3d by the technique described, and when used in conjunction with measurements of complement components and immune complexes, offers a means of investigating complement catabolism by the classical and alternative pathways.

Animals↗
PubMed2004

Selection and characterization of conditionally active promoters in Lactobacillus plantarum, using alanine racemase as a promoter probe.

This paper describes the use of the alr gene, encoding alanine racemase, as a promoter-screening tool for the identification of conditional promoters in Lactobacillus plantarum. Random fragments of the L. plantarum WCFS1 genome were cloned upstream of the promoterless alr gene of Lactococcus lactis in a low-copy-number plasmid vector. The resulting plasmid library was introduced into an L. plantarum Deltaalr strain (MD007), and 40,000 clones were selected. The genome coverage of the library was estimated to be 98%, based on nucleotide insert sequence and restriction analyses of the inserts of randomly selected clones. The library was screened for clones that were capable of complementing the D-alanine auxotroph phenotype of MD007 in media containing up to 10, 100, or 300 micro g of the competitive Alr inhibitor D-cycloserine per ml. Western blot analysis with polyclonal antibodies raised against lactococcal Alr revealed that the Alr production level required for growth increased in the presence of increasing concentrations of D-cycloserine, adding a quantitative factor to the primarily qualitative nature of the alr complementation screen. Screening of the alr complementation library for clones that could grow only in the presence of 0.8 M NaCl resulted in the identification of eight clones that upon Western blot analysis showed significantly higher Alr production under high-salt conditions than under low-salt conditions. These results established the effectiveness of the alanine racemase complementation screening method for the identification of promoters on their conditional or constitutive activity.

Alanine Racemase↗
PubMed2007

Quantitative bone mineral density assessment in malignant infantile osteopetrosis.

PURPOSE: To investigate the use of quantitative computed tomography (QCT) and dual energy absorptiometry (DXA) for assessing bone mineral density (BMD) in the evaluation of children with malignant infantile osteopetrosis (MIOP). METHODS AND PATIENTS: We retrospectively reviewed QCT- and DXA-determined BMD in six patients with infantile osteopetrosis and correlated BMD measured during the initial evaluation with patient characteristics. RESULTS: Five male and one female infant met the eligibility criteria. BMD was markedly elevated in all patients as determined by each modality, QCT or DXA. For QCT, in which age-specific normal values are known, the BMD was found to be 22.4-32.6 standard deviations above the mean. Using DXA, the estimated BMD of the lumbar spine ranged from 0.45 to 0.8 g/cm(2); children with similarly appearing bone radiographs had quite disparate BMD. While the qualitative trend of BMD among the patients was similar for both measures, the units and numerical values of BMD differed. We found no correlation between BMD results and hematopoiesis observed in the bone marrow or with visual evaluation of the radiographs. CONCLUSION: Both QCT and DXA are effective quantitative measures of BMD in children with MIOP. However, the same modality should be employed for longitudinal evaluation of a given patient. Each technique has unique advantages and disadvantages and may complement one another in the evaluation of MIOP or other bone disorders. Quantitative assessment of BMD in children with MIOP will likely serve to further characterize this disease.

Absorptiometry, Photon↗
PubMed1987

Quantitative immunocytochemical characterization of mononuclear phagocytes. I. Monoblasts, promonocytes, monocytes, and peritoneal and alveolar macrophages.

The purpose of the present study was to compare the phenotype of tissue macrophages with that of their precursors in the bone marrow and blood. The phenotype was determined on the basis of the quantitative binding of monoclonal antibodies to cell-surface antigens (antigen F4/80, complement receptor III, Fc receptor II, Ia antigen, common leukocyte antigen, and Mac-2 and Mac-3 antigens) on individual mononuclear phagocytes. Monoclonal antibody binding to cells, detected by the biotin-avidin immunoperoxidase procedure, was quantitated by cytophotometric determination of the amount of enzyme reaction product on cells. The results of this quantitation are expressed as the median of the specific absorbance per unit of cell-surface area (0.25 micron2) and per cell. Shortly after collection of the mononuclear phagocytes, binding of all monoclonal antibodies except those directed against the common leukocyte and Mac-2 antigens to peritoneal macrophages was enhanced compared with binding to blood monocytes; for alveolar macrophages we found reduced binding of monoclonal antibodies F4/80 and M1/70 (complement receptor III) and enhanced binding of monoclonal antibodies with specificity for the common leukocyte antigen and Mac-2 and Mac-3 antigens. The results obtained with cultured mononuclear phagocytes show that during the development from monoblast to tissue macrophages, monoclonal antibody binding to the various types of mononuclear phagocyte, expressed per unit of cell-surface area, was not significantly altered except that of M3/38 (Mac-2 antigen) to peritoneal macrophages and that of F4/80 and M1/70 (complement receptor III) to alveolar macrophages. Expressed on a per cell basis, the results show an increase in the binding of all monoclonal antibodies except those directed against the Fc receptor II and Mac-3 antigen during the development from promonocytes to peritoneal macrophages; binding of most monoclonal antibodies to alveolar macrophages was considerably lower than that to blood monocytes. It is concluded that the expression of the various cell-surface antigens alters during mononuclear phagocyte differentiation. The expression changed also during culture, although distinct patterns of alteration could not be distinguished.

Animals↗
PubMed2006

Gene array analysis of a rat model of liver transplant tolerance identifies increased complement C3 and the STAT-1/IRF-1 pathway during tolerance induction.

This study aimed to define the molecular mechanism during induction of spontaneous liver transplant tolerance using microarrays and to focus on molecular pathways associated with tolerance by meta-analysis with published studies. The differences in the early immune response between PVG to DA liver transplant recipients that are spontaneously tolerant (TOL) and PVG to Lewis liver transplants that reject (REJ) were examined. Spleens from TOL and REJ on days 1 and 3 were compared by 2 color microarray. Forty-six of 199 genes differentially expressed between TOL and REJ had an immunological function. More immune genes were increased in TOL vs. REJ on day 1, including STAT-1, IRF-1 and complement C3. Differential expression of selected genes was confirmed by quantitative RT-PCR. The results were compared to two published high-throughput studies of rat liver transplant tolerance and showed that C3 was increased in all three models, while STAT-1 and IRF-1 were increased in two models. The early increases in immune genes in TOL confirmed previous reports of an active early immune response in TOL. In conclusion, the increase in STAT-1, IRF-1 and complement component C3 in several models of liver transplant tolerance suggests that the STAT-1/IRF-1 apoptotic pathway and C3 may be involved in the tolerogenic mechanism.

Animals↗
PubMedDate not supplied

[Significance of immune complexes in children with severe hemophilia A in substitution treatment with factor VIII concentrates].

Sera and EDTA-Plasma of patients with severe Haemophilia A were analysed for immune complexes and the hemolytic activity of complement in relation to Factor VIII replacement, in order to confirm or possibly exclude a relationship to allergic reactions. Immune complexes were isolated by PEG precipitation and quantitated. In addition a solid phase ELISA assay was used to detect complement-binding complexes. Total hemolytic complement activity of the classical and the alternate pathway was measured in addition to the C3 splitproduct C3d. The results obtained from 12 patients with severe Haemophilia A showed slightly increased immune complex titers, no changes of the immune complex levels during Factor VIII replacement and no alteration of the complement system following the infusions. One patient developed an allergic reaction without evidence of complement activation.

Adolescent↗
PubMed1983

Platelet-associated complement C3 in thrombocytopenic states.

Platelet-associated complement components C3, C3d and C4 (PAC3, PAC3d, PAC4) were quantitated by a modification of the platelet radioactive anti-IgG test using highly purified C3 antibodies in 74 patients with immune thrombocytopenia (ITP), 26 patients with presumed nonimmune thrombocytopenia (NTP), and 114 normal individuals. Elevated PAC3 levels were found in 26 out of 74 patients with ITP (35%) and in 10 out of 26 patients with NTP. Although the percentage of elevated PAC3 values was higher in thrombocytopenic patients than in nonthrombocytopenic patients, no statistically significant correlation existed between platelet counts and PAC3 levels, neither for ITP nor for NTP. However, such a relationship was demonstrable between PAC3 and platelet-associated IgG, both for ITP (P less than 0.05) as well as NTP patients (P less than 0.001). We conclude that elevated PAC3 values are not restricted to immune thrombocytopenias. Quantitative differences of PAC3 between ITP and NTP patients suggest that part of the PAC3 is immunologically mediated and has a role in the pathogenesis of autoimmune thrombocytopenias.

Adult↗
PubMed2000

Efficient marker-based recurrent selection for multiple quantitative trait loci.

We studied the efficiency of recurrent selection based solely on marker genotypes (marker-based selection), in order to increase favourable allele frequency at 50 previously detected quantitative trait loci (QTLs). Two selection procedures were investigated, using computer simulations: (1) Truncation Selection (MTS), in which individuals are ranked based on marker score, and best individuals are selected for recombination; and (2) QTL Complementation Selection (QCS), in which individuals are selected such that their QTL composition complements those individuals already selected. Provided QTL locations are accurate, marker-based selection with a population size of 200 was very effective in rapidly increasing frequencies of favourable QTL alleles. QCS methods were more effective than MTS for improving the mean frequency and fixation of favourable QTL alleles. Marker-based selection was not very sensitive to a reduction in population size, and appears valuable to optimize the use of molecular markers in recurrent selection programmes.

Alleles↗
PubMed1975

Immunologic aspects of term pregnancy toxemia. A study of immunoglobins and complement.

IgA, IgG, and IgM were measured by the single radial immunodiffusion method in umbilical cord and maternal sera in toxemia patients and in normal term pregnant cases. Complement (C' 3) levels were determined only in maternal sera. Quantitation of IgA, IgG, and IgM was performed in concentrated serial urine samples from three eclamptic and three normal control patients. Results show that there are lower IgG and IgM levels in the sera of mothers with toxemia of pregnancy; however, paradoxically higher IgM levels were detected in cord sera from their newborn infants without evidence of placental leakage or increased neonatal infection. The finding of the low IgG and IgM levels appears to be due in part to immunoglobin loss into the urine. The unchanged complement levels in toxemia, in this study, are not suggestive of an active immunologic process, but the high IgM in the newborn infants of toxemic mothers is unexplained and may represent active immunologic disease. Clearly, investigations of tissue binding and the formation of immunoglobin complexes should be carried out to better explain these abnormal findings.

Blood Protein Electrophoresis↗
PubMed2001

Hereditary angioedema: a broad review for clinicians.

Hereditary angioedema (HAE) is an autosomal dominant disease that afflicts 1 in 10,000 to 1 in 150,000 persons; HAE has been reported in all races, and no sex predominance has been found. It manifests as recurrent attacks of intense, massive, localized edema without concomitant pruritus, often resulting from one of several known triggers. However, attacks can occur in the absence of any identifiable initiating event. Historically, 2 types of HAE have been described. However, a variant, possibly X-linked, inherited angioedema has recently been described, and tentatively it has been named "type 3" HAE. Signs and symptoms are identical in all types of HAE. Skin and visceral organs may be involved by the typically massive local edema. The most commonly involved viscera are the respiratory and gastrointestinal systems. Involvement of the upper airways can result in severe life-threatening symptoms, including the risk of asphyxiation, unless appropriate interventions are taken. Quantitative and functional analyses of C1 esterase inhibitor and complement components C4 and C1q should be performed when HAE is suspected. Acute exacerbations of the disease should be treated with intravenous purified C1 esterase inhibitor concentrate, where available. Intravenous administration of fresh frozen plasma is also useful in acute HAE; however, it occasionally exacerbates symptoms. Corticosteroids, antihistamines, and epinephrine can be useful adjuncts but typically are not efficacious in aborting acute attacks. Prophylactic management involves long-term use of attenuated androgens or antifibrinolytic agents. Clinicians should keep this disorder in their differential diagnosis of unexplained, episodic cutaneous angioedema or abdominal pain.

Acute Disease↗
PubMed2004

Autoradiographic distribution of serotonin transporters and receptor subtypes in human brain.

Several neurochemical in vitro and in vivo imaging studies have been aimed at characterizing the localization of serotonin receptors and transporters in the human brain. In this study, a detailed comparison of the distribution of a number of 5-HT receptor subtypes and the 5-HT transporter was carried out in vitro using human postmortem brain tissue. Anatomically adjacent whole hemisphere sections were incubated with specific radioligands for the 5-HT(1A), 5-HT(1B), 5-HT(2A), 5-HT(4) receptors and the 5-HT transporter. The autoradiograms revealed different laminar and regional distribution patterns in the isocortex, where 5-HT(1A) and 5-HT(4) receptor binding showed highest densities in superficial layers and 5-HT(2A) receptor binding was most abundant in middle layers. In cortical regions, 5-HT transporters were concentrated to several limbic lobe structures (posterior uncus, entorhinal, cingulate, insular and temporal polar regions). 5-HT(1A) receptor densities were also high in limbic cortical regions (hippocampus, posterior entorhinal cortex, and subcallosal area) compared to the isocortex. Subregionally different distribution patterns were observed in the basal ganglia with a trend toward higher levels in ventral striatal (5-HT(1B) receptors) and pallidal (5-HT transporters and 5-HT(1B) receptors) regions. The localization in regions belonging to limbic cortico-striato-pallido-thalamic circuits is in line with the documented role of 5-HT in modulation of mood and emotion, and the suggested involvement of this system in pathophysiology of various psychiatric disorders. The qualitative and quantitative information reported in this study might provide important complements to in vivo neuroimaging studies of the 5-HT system.

Autoradiography↗
PubMed1996

Tissue-level biomarkers and biological effect of mercury on sentinel slugs, Arion ater.

The present investigation was to determine the extent to which mercury (Hg) provokes measurable effects on the structure of the digestive gland of slugs as well as to relate the extent of these effects to the cell and tissue distribution of Hg. For this purpose, slugs (Arion ater) received various dietary concentrations of Hg (from 0 to 1,000 microg Hg/g food) as chloride for 30 days and were histologically examined every third day. Autometallography was used to demonstrate Hg as black silver deposits (BSD) in paraffin sections. The lysosomes and residual bodies of digestive cells resulted to be the major accumulation sites. In addition, Hg was also evidenced in lipofuscine granules of vacuoles in excretory cells but, however, it was rarely observed within calcium cells. Generally, the extent of BSD increased with dietary Hg concentration and exposure time but, however, it became significant lowered after exposure to 1,000 microg Hg/g food for 30 when the digestive epithelium appeared almost devoid of digestive cells. On the other hand, significant changes were recorded in the quantitative structure of digestive tubules. Mean Epithelial Thickness (MET), Mean Luminal Radius (MLR) and Mean Diverticular Radius (MDR) were recorded as measures of the sublethal biological effect of Hg. MET, MLR/MET and MET/MDR were affected by Hg concentration (C), exposure time (T) and CxT interaction, changes in MET, MLR, MLR/MET and MET/MDR being explained by regression models after logarithmic transformation of the data. In order to explain the nature of the changes in the quantitative structure of the digestive tubules this investigation was complemented with qualitative histological observations. According to them, the excretory activity in digestive cells was initially enhanced. Afterwards, the relative numbers of digestive cells declined until the extreme cases of exposure to 1, 000 microg Hg/g for 27 to 30 days in which the digestive epithelium was mostly comprised of calcium and excretory cells. Concomitantly, some changes took place in blood vessels where Leydig cells became disrupted and the connective tissue layers thickened. Finally, it is suggested to use slugs in soil quality assessment as sentinel organisms ("Slug Watch") in which biomarkers of exposure to metallic pollutants and of biological effect are recorded.

Animals↗
PubMed1979

A microplate immunoenzyme assay for anti-influenza antibodies.

An enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase is described for the detection and quantitation of anti-influenza virus antibodies. Compared with complement fixation and hemagglutination inhibition tests, ELISA is far superior with respect to sensitivity and reliability. Non-specific viral inhibitors present in sera do not affect the titer in ELISA. Its sensitivity, close to that of radioimmunoassay permits detection of small amounts of antibodies in pulmonary secretions and supernatants from in vitro spleen cell cultures.

Animals↗