Reactive lysis: the complement-mediated lysis of unsensitized cells. I. The characterization of the indicator factor and its identification as C7.

This paper describes the characteristics of the indicator factor (I) which takes part in reactive hemolysis and its identification as the seventh component of complement. I was shown to be a beta globulin with a sediment coefficient of 5.7S and a molecular weight of about 140,000. Experiments on the depletion of I activity with anti-I antiserum or with activated R euglobulin showed that I was a late acting complement component necessary for the lysis of cells after the EAC142 stage. Complement component analysis of purified I fractions excluded all known components except C7. The physicochemical characteristics of I are compatible with published data on C7. The method of quantitation described represents a convenient method of testing for C7.

Reactive lysis: the complement-mediated lysis of unsensitized cells. II. The characterization of activated reactor as C56 and the participation of C8 and C9.

It has been shown that the "activated reactor" that is produced in certain human sera by complement activation is a stable complex of the fifth and sixth component of complement (C56). On interaction with C7, the indicator factor, a complex C567 is formed which for a short time (half-life less than 1 min) has an activated binding site and can attach itself to normal red cell membranes, conferring on them the hemolytic properties of the "heat stable" complement intermediate EC 1 approximately 7, the capacity to be lysed by C8 and C9. These cells have neither antibody nor the complement components up to C3 bound on them. The binding site-activated C567c-can similarly bind to other hydrophobic surfaces, including agarose gel where it forms a "stainable line". If the complex is not bound to a surface, the binding site decays and the resulting complex will no longer give rise to lysis. However it will still inactivate C8 and C9 in solution. The sera that can generate activated reactor apparently do so because they have an excess of C5 and C6, compared to their content of C7. The phenomenon of reactive lysis thus represents complement-mediated lysis of unsensitized cells initiated at the C5 stage by a stable complex (C56) which was generated by complement activation at a distance. The immunochemistry of the phenomenon is described and some of its implications discussed.

Body rate decoupling using haltere mid-stroke measurements for inertial flight stabilization in Diptera.

Halteres, the modified rear wings of Diptera, have long been recognized as sensory organs necessary for basic flight stability. These organs, which act as vibrating structure gyroscopes, are known to sense strains proportional to Coriolis accelerations. While compensatory responses have been demonstrated that indicate the ability of insects to distinguish all components of the body rate vector, the specific mechanism by which the halteres are able to decouple the body rates has not been clearly understood. The research documented in this report describes a potential mechanism, using averaged strain and strain rate at the center of the haltere stroke, to decouple the inertial rate components. Through dynamic simulation of a nonlinear model of the haltere 3-dimensional trajectory, this straightforward method was demonstrated to provide an accurate means of generating signals that are proportional to three orthogonal body rate components. Errors associated with residual nonlinearity and rate-coupling were quantified for a bilaterally reconstructed body rate vector over a full range of pitch and yaw rates and two roll rate conditions. Models that are compatible with insect physiology are proposed for performing necessary signal averaging and bilateral processing.

Role of migratory birds in introduction and range expansion of Ixodes scapularis ticks and of Borrelia burgdorferi and Anaplasma phagocytophilum in Canada.

During the spring in 2005 and 2006, 39,095 northward-migrating land birds were captured at 12 bird observatories in eastern Canada to investigate the role of migratory birds in northward range expansion of Lyme borreliosis, human granulocytic anaplasmosis, and their tick vector, Ixodes scapularis. The prevalence of birds carrying I. scapularis ticks (mostly nymphs) was 0.35% (95% confidence interval [CI] = 0.30 to 0.42), but a nested study by experienced observers suggested a more realistic infestation prevalence of 2.2% (95% CI = 1.18 to 3.73). The mean infestation intensity was 1.66 per bird. Overall, 15.4% of I. scapularis nymphs (95% CI = 10.7 to 20.9) were PCR positive for Borrelia burgdorferi, but only 8% (95% CI = 3.8 to 15.1) were positive when excluding nymphs collected at Long Point, Ontario, where B. burgdorferi is endemic. A wide range of ospC and rrs-rrl intergenic spacer alleles of B. burgdorferi were identified in infected ticks, including those associated with disseminated Lyme disease and alleles that are rare in the northeastern United States. Overall, 1.4[corrected]% (95% CI = 0.3 [corrected] to 0.41) of I. scapularis nymphs were PCR positive for Anaplasma phagocytophilum. We estimate that migratory birds disperse 50 million to 175 million I. scapularis ticks across Canada each spring, implicating migratory birds as possibly significant in I. scapularis range expansion in Canada. However, infrequent larvae and the low infection prevalence in ticks carried by the birds raise questions as to how B. burgdorferi and A. phagocytophilum become endemic in any tick populations established by bird-transported ticks.

Analysis of RUNX1 binding site and RAPTOR polymorphisms in psoriasis: no evidence for association despite adequate power and evidence for linkage.

BACKGROUND: A previous study identified two peaks of allelic association between psoriasis and single nucleotide polymorphisms (SNPs) mapping to distal chromosome 17q, including a disease associated SNP that leads to loss of a RUNX1 transcription factor binding site, and additional SNPs in the third intron of the RAPTOR gene. Another study found an association with SNPs in the RAPTOR gene, but not with the RUNX1 binding site polymorphism. METHODS: In an effort to confirm these observations, we genotyped 579 pedigrees containing 1285 affected individuals for three SNPs immediately flanking and including the RUNX1 binding site, and for three SNPs in the RAPTOR gene. RESULTS: Here we report further evidence for linkage to distal chromosome 17q, with a linkage peak mapping 1.7 cM distal to the RUNX1 binding site (logarithm of the odds 2.26 to 2.73, depending upon statistic used). However, we found no evidence for association to individual SNPs or haplotypes in either of the previously identified peaks of association. Power analysis demonstrated 80% power to detect significant association at genotype relative risks of 1.2 (additive and multiplicative models) to 1.5 (dominant and recessive models) for the RUNX1 binding site, and 1.3 to 1.4 for the RAPTOR locus under all models except dominant. CONCLUSIONS: Our data provide no support for the previously identified RUNX1 binding site or for the RAPTOR locus as genetic determinants of psoriasis, despite evidence for linkage of psoriasis to distal chromosome 17q.

Ixodes scapularis ticks collected by passive surveillance in Canada: analysis of geographic distribution and infection with Lyme borreliosis agent Borrelia burgdorferi.

Passive surveillance for the occurrence of the tick Ixodes scapularis Say (1821) and their infection with the Lyme borreliosis spirochaetes Borrelia burgdorferi s.l. has taken place in Canada since early 1990. Ticks have been submitted from members of the public, veterinarians, and medical practitioners to provincial, federal, and university laboratories for identification, and the data have been collated and B. burgdorferi detected at the National Microbiology Laboratory. The locations of collection of 2,319 submitted I. scapularis were mapped, and we investigated potential risk factors for I. scapularis occurrence (in Quebec as a case study) by using regression analysis and spatial statistics. Ticks were submitted from all provinces east of Alberta, most from areas where resident I. scapularis populations are unknown. Most were adult ticks and were collected in spring and autumn. In southern Québec, risk factors for tick occurrence were lower latitude and remote-sensed indices for land cover with woodland. B. burgdorferi infection, identified by conventional and molecular methods, was detected in 12.5% of 1,816 ticks, including 10.1% of the 256 ticks that were collected from humans and tested. Our study suggests that B. burgdorferi-infected I. scapularis can be found over a wide geographic range in Canada, although most may be adventitious ticks carried from endemic areas in the United States and Canada by migrating birds. The risk of Lyme borreliosis in Canada may therefore be mostly low but more geographically widespread than previously suspected.

Investigation of ground level and remote-sensed data for habitat classification and prediction of survival of Ixodes scapularis in habitats of southeastern Canada.

In southeastern Canada, most populations of Ixodes scapularis Say, the Lyme disease vector, occur in Carolinian forests. Climate change projections suggest a northward range expansion of I. scapularis this century, but it is unclear whether more northerly habitats are suitable for I. scapularis survival. In this study, we assessed the suitability of woodlands of the Lower Great Lakes/St. Lawrence Plain region for I. scapularis by comparing tick egg survival in four different woodlands. Woodlands where I. scapularis are established, and sand dune where I. scapularis do not survive, served as positive and negative control sites, respectively. At two woodland sites, egg survival was the same as at the positive control site, but at two of the sites survival was significantly less than either the positive control site, or one of the other test sites. Egg survival in all woodland sites was significantly higher than in the sand dune site. Ground level habitat classification discriminated among woodlands in which tick survival differed. The likelihood that I. scapularis populations could persist in the different habitats, as deduced using a population model of I. scapularis, was significantly associated with variations in Landsat 7 ETM+ data (normalized difference vegetation index [NDVI] and Tasselled Cap indices). The NDVI index predicted habitat suitability at Long Point, Ontario, with high sensitivity but moderate specificity. Our study suggests that I. scapularis populations could establish in more northerly woodland types than those in which they currently exist. Suitable habitats may be detected by ground-level habitat classification, and remote-sensed data may assist this process.

Network analysis of Danish cattle industry trade patterns as an evaluation of risk potential for disease spread.

Trade patterns of animal movements in a specific industry are complex and difficult to study because there are many stakeholders, premises that are heterogeneously spread over the country, and a highly dynamic flow of animals exists among them. The Danish cattle industry was defined as a network of animal movements and graph theory was used to analyse the movements of cattle within this network. A premise was defined as a farm, an abattoir or a market. These premises constituted the network nodes in the graph and the animal movements between them were the links. In this framework, each premise had a sub-network of other premises to which it was linked by these animal movements. If no movement of animals were registered for a specific farm, then the sub-network for that premise consisted of only that premise. Otherwise, the sub-network linked the premise of interest to all premises from which and to which animals were moved, as long as there was a path linking animal movements to that specific premise. This approach allowed visualization and analyses of four levels of organization that existed in Denmark animal registers: (1) the animal that was moved, (2) the movements of all animals between two premises, (3) the specific premise network, and (4) the overall industry network. When contagious animals are moved from one premise to another, then to a third and so forth, these movements create a path for potential transfer of pathogens. The paths within which pathogens are present identify the transmission risks. A network of animal movements should provide information about pathogen transmission and disease spread. The network of the Danish cattle industry network was a directed scale-free graph (the direction of a movement was known), with an in-degree power of 2 an out-degree power of 1.46, consisted of 29,999 nodes, and 130,265 movements during a 6-month period. The in clustering coefficient was calculated to be 0.52 for the inward direction (movement to), while it was 0.02 for the outward direction (movement from). In Denmark, the cattle movements between premises demonstrated a large degree of heterogeneity. This heterogeneity in movements between farms should be used to evaluate the risk potential of disease transmission for each premise and must be considered when modelling disease spread between premises. The objective of this research was to describe the network of animal movements and not just the animal movements per se.

Plasmapheresis in hereditary angioneurotic edema and systemic lupus erythematosus.

A patient had hereditary angioneurotic edema coincident with systemic lupus erythematosus. This condition resulted in loss of hair, arthralgia, leukoplakia, collitis, and a nephrotic syndrome due to proliferative glomerulonephritis. The renal lesion was resistant to treatment with high-dose prednisolone and cyclophosphamide; however, sustained remission occurred after plasmapheresis. The patient has remained well for over two years since this procedure.

Characterization of a non-functional form of C1q found in patients with a genetically linked deficiency of C1q activity.

A genetically defective form of C1q was purified from the sera of patients suffering from an immune complex related disease and who were homozygous for the defect. The defective C1q was haemolytically inactive and did not bind to immune aggregates or IgG-Sepharose. It showed the following similarities to the normal C1q molecule: a high glycine content and the presence of hydroxyproline and hydroxylysine; subunits with apparent mol. wts of 70,000 and 56,000, when examined by SDS-polyacrylamide gel electrophoresis under non-reducing conditions; preferential incorporation of 125I-label into only one of the types of chain present in the molecule, in a manner similar to that found for the C-chain of normal C1q. However, the defective molecule had an apparent mol. wt of approximately 155,000 in non-dissociating conditions, which is approximately one-third of the mol. wt of the normal molecule. Also, the material in the defective molecule preparation which corresponded, on the basis of mol. wt, to the disulphide-linked A-chain-B-chain dimer of normal C1q differed from that found in the normal molecule in that it did not appear to be sensitive to reducing agents. Collagenase and pepsin treatment of specific immunoprecipitates containing the radiolabelled defective molecule indicated that it is, like the normal molecule, composed of collagenous and non-collagenous domains.

Subclass distribution of IgG autoantibodies in bullous pemphigoid.

The distribution of IgG subclasses in the antibasement membrane zone autoantibody of pemphigoid in skin and serum was analyzed by use of monoclonal antibodies to human IgG subclasses. The predominant subclass was IgG4 which was present in 23 of 24 skin biopsies, IgG1 was next and IgG3 was found only occasionally. In 3 of 24 biopsies IgG4 was the only IgG subclass detected, C3 was absent in 2 of these, the third contained IgM and C3. Serum autoantibodies were similarly analyzed by indirect immunofluorescence (IIF) when again IgG4 autoantibody was the dominant subclass. No IgE autoantibody was detected by IIF.

Measurement of functional C2--a method for clinical laboratories.

A method is described which measures functional C2 in human serum using the readily available reagents, sensitised sheep erythrocytes, zymosan-treated normal human serum, and guinea pig serum.

Evaluation of an enzyme immunoassay for total IgE on a semi-automated analyser. A multicentre study.

Total serum IgE was measured by a semi-automated enzyme immunoassay on sera from normal and disease groups. Data from this investigation was analyzed in respect of precision, linearity, sensitivity and correlation with other test methods. Using human serum pools having values between 7 and 480 IU/ml, the intra-assay coefficient of variation ranged from 3.3 to 14.6% with an arithmetic mean of 6%. The inter-assay coefficient of variation on commercially supplied control sera ranged from 4.4 to 14.2%. In addition, tests were carried out on serially diluted samples to assess the linearity of the method, and on sera with IgE levels of less than 5 IU/ml in order to assess its sensitivity. It was shown that the technique being assessed was unaffected by the presence of lipid or haemoglobin or by the addition of bilirubin or any one of 46 commonly prescribed drugs each at double its toxic dose. There was good correlation between the semi-automated enzyme immunoassay technique and four other methods used during this study. This technique exhibits excellent specificity, reproducibility and a sensitivity well within clinical demands.

Purification of human blood eosinophils by negative selection using immunomagnetic beads.

A simple method for isolating highly purified eosinophils from human blood is described. Buffy coats from normal individuals (eosinophil counts less than 0.4 x 10(9)/litre) were centrifuged through a two layer Percoll density gradient, to produce a granulocyte fraction containing neutrophils and eosinophils. Neutrophils were extracted from this fraction using a monoclonal antibody (CLB FcR gran 1) against CD 16 (Fc gamma R III) in a direct or indirect selection procedure using immunomagnetic beads (Dynabeads). This negative immunoselection produced eosinophils of greater purity and with a superior capacity to mount a respiratory burst than eosinophils isolated by a method employing metrizamide.

Loss of bactericidal capacity of long-chain quaternary ammonium compounds with protein at lowered temperature.

Amphiphilic betaine esters are quaternary ammonium compounds (QACs) with rapid microbicidal effect, which spontaneously hydrolyze into nontoxic products. thus being referred to as soft antimicrobial agents. The bactericidal effect of 1-decyl (B10), 1-dodecyl (B12), and 1-tetradecyl (B14) betaine esters on Salmonella typhimurium was strongly influenced by temperature, pH and length of hydrocarbon chain. At pH 6.0, presence of 1.5 mM (10% w/v) BSA raised the concentration of B14 for 99% killing (BC2) from 0.006 mM to 1.8 mM. There was a stoichiometric relationship between concentration of BSA and BC2 of B14, indicating that one molecule of B14 was bound per BSA molecule when 99% killing was achieved. When the temperature was lowered to 0 degrees C only minor killing was seen in 1.5 mM BSA at the highest concentration of B14 tested, 57 mM. With B10 at 30 degrees C and pH 6.0, the presence of 1.5 mM BSA raised the bactericidal concentration (BC2) from 0.69 mM to 4.1 mM, and at 0 degrees C and 1.5 mM BSA the BC2 was 11 mM. Thus, the impairment caused of the bactericidal effect of B10 by BSA and lower temperature was less than for B14, since B14 is much more active than B10 at 30 degrees C in the absence of BSA, somewhat more active than B10 at 30 degrees C in the presence of 1.5 mM BSA, and much less active than B10 at 0 degrees C in the presence of BSA. B12 showed properties intermediate between B10 and B14. Lowered pH reduced the bactericidal effect particularly when reduced from pH 5.0 to 4.0 with B10. In the presence of 1.5 mM BSA, the bactericidal effect of 1-dodecyl (DTAB) and 1-hexadecyl (CTAB) trimethylammonium bromide decreased in the same manner as for B10 and B14, respectively. Increasing the time of incubation at 0 degrees C to 50 min, a 99% killing effect was seen with 17 mM CTAB, whereas the same killing effect was reached in 8 min with 17 mM DTAB. Binding of [3H]CTAB to S. typhimurium was also reduced at 0 degrees C in the presence of BSA. Thus, in the presence of 1.5 mM BSA, QACs with the longer hydrocarbon chain were most efficient at 30 degrees C, whereas at 0 degrees C those with the shorter hydrocarbon chain were most active. Consequently, QACs with shorter tails should be used for disinfection in the presence of proteins at lower temperatures.

The effect of hydrocarbon chain length, pH, and temperature on the binding and bactericidal effect of amphiphilic betaine esters on Salmonella typhimurium.

Amphiphilic betaine esters are quaternary ammonium compounds (QAC) with rapid microbicidal action. They are often labeled 'soft antimicrobial agents', since the compounds hydrolyze spontaneously into betaine and fatty alcohols, thus not only losing their surface active properties and toxicity but also becoming amenable to metabolic use. The present results show that the bactericidal effects of 1-decyl (B10), 1-dodecyl (B12), and 1-tetradecyl (B14) betaine esters on Salmonella typhimurium 395 MS decreased with decreasing hydrocarbon chain lengths, decreased at pH below neutral, and were lower at 0 degrees C that at 30 degrees C. At least part of the decreased effect at pH 4.0 as compared to pH 6.0 can be explained by reduced binding. However, reduced binding cannot explain the decrease in the microbicidal effect at 0 degrees C since the binding of B 14 was the same at 0 degrees C and 30 degrees C although 10-30 times higher concentrations were required at 0 degrees C to achieve the same microbicidal effect as at 30 degrees C. Neither can differences in binding explain the great differences seen in microbicidal effect between QAC with different chain lengths. It is proposed that the membrane deformation resulting in killing of S. typhimurium is more efficiently achieved with QAC with longer hydrocarbon chains and that reduced fluidity of the outer membrane of the bacteria at lower temperatures antagonizes the bactericidal effect. Charge interaction seems to be more important for the binding and bactericidal effect for the QAC with shorter hydrocarbon chains. The different effects of pH, temperature, and hydrocarbon chain length on binding, bactericidal effect, and hydrolysis have to be taken into account when optimizing disinfection and the subsequent elimination of disinfectants.

A method for the estimation of the activity of the inhibitor of the first component of complement.

The activated first component of complement (CI) possesses an esterase activity in vitro which will hydrolyse an ester of tyrosine to release H+. The activity of the serum inhibitor of C1 esterase may be measured by monitoring its ability to prevent H+ release under standard conditions. This paper describes a method of measuring such activity, monitoring H+ release by the use of either an acid base indicator of pH meter.

Paraprotein estimation: a comparison of immunochemical and densitometric techniques.

Paraproteins have been estimated by both immunochemical methods and densitometric analysis of electrophoretic strips. Correlation between the results obtained by these two methods, although generally good for assessing trends, varied between patients.

Severe selective IgM deficiency.

Among the primary antibody deficiency syndromes, severe selective IgM deficiency (also previously known as type V dysgammaglobulinaemia) is rare, and the majority of previous reports have indicated a fatal outcome. Three adult patients who were found to have a persistently low serum IgM are described. This deficiency was not obviously related to their presenting illness; in two of the patients, who were investigated in detail, it appeared to be of no immediate consequence.

A simple, rapid ELISA method for the detection of DNA antibodies.

Employing an enzyme-linked immunosorbent assay (ELISA) technique the serum antibodies against native (double stranded) and denatured (single stranded) deoxyribonucleic acid (DNA) have been measured in various disease groups and a group of blood donor sera. The ELISA method has been compared with a radioimmunoassay method using native (double stranded) DNA is substrate antigen and a latex-fixation technique using particles coated with soluble deoxyribonucleoprotein (SNP). It is concluded that ELISA offers an economic and reliable alternative to isotope techniques for the assessment of antibody content in systemic lupus erythematosus (SLE) and related disease states for the clinical laboratory.