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PubMed2002

Use of recombinase activation gene-2 deficient mice to ascertain the role of cellular and humoral immune responses in the development of chronic rejection.

INTRODUCTION: Given its multifactorial etiology, the relative contribution of anti-donor cellular and humoral immune responses in the pathogenesis of chronic rejection is as yet ambiguous. We hypothesized that alloreactive T and B cells play a seminal role in the development of this lesion. METHODS: To address this hypothesis, RAG-2(-/-) mice were used as donors and recipients in a well-established murine model of aortic transplantation. Grafts were transplanted across the following groups: Group I: C3H --> C3H; Group II: Wild-type [WT] 129Sv (H-2(b)) --> C3H (H-2(k)); Group III: C3H --> WT 129Sv; Group IV: 129SvEv RAG-2(-/-) --> C3H; and Group V: C3H --> 129SvEv RAG-2(-/-). Grafts were harvested at d40 to 146 post-transplantation for morphologic and immunohistochemical analyses and semi-quantitative RT-PCR was employed to evaluate the intragraft mRNA expression of various immune mediators. Mixed lymphocyte reaction and complement-mediated alloantibody cytotoxicity assays were performed to determine anti-donor proliferative and humoral responses, respectively. RESULTS: Unlike that across the syngeneic combination (Group I), marked intimal thickening with corresponding luminal narrowing was observed in the majority of the aortic allografts (Groups II-IV). On the contrary, the morphology of C3H aortic allografts harvested from the majority of the RAG-2(-/-) was remarkably preserved. Correspondingly, anti-donor proliferative and humoral immune responses were undetectable in C3H --> RAG-2(-/-) recipients as was the intragraft mRNA expression of the Th(1) and the Th(2)-type cytokines. CONCLUSIONS: Taken together, these data suggest that in this murine model of aortic allotransplantation, donor-specific cellular and humoral responses play a dominant role in the initiation and perpetuation of chronic rejection.

Animals↗
PubMed2001

Defining the catalytic metal ion interactions in the Tetrahymena ribozyme reaction.

Divalent metal ions play a crucial role in catalysis by many RNA and protein enzymes that carry out phosphoryl transfer reactions, and defining their interactions with substrates is critical for understanding the mechanism of biological phosphoryl transfer. Although a vast amount of structural work has identified metal ions bound at the active site of many phosphoryl transfer enzymes, the number of functional metal ions and the full complement of their catalytic interactions remain to be defined for any RNA or protein enzyme. Previously, thiophilic metal ion rescue and quantitative functional analyses identified the interactions of three active site metal ions with the 3'- and 2'-substrate atoms of the Tetrahymena group I ribozyme. We have now extended these approaches to probe the metal ion interactions with the nonbridging pro-S(P) oxygen of the reactive phosphoryl group. The results of this study combined with previous mechanistic work provide evidence for a novel assembly of catalytic interactions involving three active site metal ions. One metal ion coordinates the 3'-departing oxygen of the oligonucleotide substrate and the pro-S(P) oxygen of the reactive phosphoryl group; another metal ion coordinates the attacking 3'-oxygen of the guanosine nucleophile; a third metal ion bridges the 2'-hydroxyl of guanosine and the pro-S(P) oxygen of the reactive phosphoryl group. These results for the first time define a complete set of catalytic metal ion/substrate interactions for an RNA or protein enzyme catalyzing phosphoryl transfer.

Animals↗
PubMed1976

Quantitation of serum antibodies to surface antigens of Neisseria gonorrhoeae with radiolabeled protein A of Staphylococcus aureus.

Antibodies in human sera against surface antigens of Neisseria gonorrhoeae were detected with use of 125I-labeled protein A from Staphylococcus aureus. Serum was allowed to react with a suspension of whole gonococci, and the antibodies attached to the bacteria were quantitated with protein A, which reacts with the Fc fragment of IgG. Tests with five human sera with a gonococcoal complement-fixation titer of greater than or equal to 1:30 revealed no difference between use of freshly isolated gonococci and use of strains subcultured daily on artificial medium for 10 years. Antibodies cross-reacting with N. gonorrhoeae and Neisseria meningitidis were found in human sera. The results of the test with labeled protein A varied with the serum titer of complement-fixing antibody to N. gonorrhoeae. Acute and convalescent sera from six of seven patients, including one with disseminated gonococcal infection, showed significantly larger differences in antibodies to N. gonorrhoeae than did sera of women without evidence of genital infection. Complement-fixation titers changed significantly in only three of the six patients.

Adolescent↗
PubMed1995

A genetic and physical map of bovine chromosome 3.

This paper reports a map of nine polymorphic microsatellite markers previously assigned to bovine chromosome 3 (BTA3) by somatic cell genetics. The linkage group covers 101 cM on the chromosome with an average intermarker distance of 13.9 cM. One marker (INRA200) was isolated from a peak of flow sorted chromosomes 2 and 3. Another marker (INRA197) was derived from a cosmid. The localization of the cosmid by in situ hybridization enabled the orientation of the linkage group on BTA3. Markers were relatively evenly spaced and consequently can be used to complement other mapping data about this chromosome. This establishes a framework of polymorphic markers that can be used to search for quantitative trait loci (QTL).

Animals↗
PubMed2006

Development of non-agglutination microarray blood grouping.

Microarray technology provides an opportunity to monitor multiple parameters simultaneously. High-throughput applications such as blood donation screening could greatly benefit from performing various tests on a single testing platform. Blood grouping represents one part of the donation testing complementing the screening for blood-borne pathogens. Blood group serology traditionally exploited agglutination as the detection method. In this investigation, we have adapted blood grouping reactions to a solid-phase microarray substrate in a non-agglutination reaction format as an initial step in the development of a combined microarray testing platform. We have investigated immobilization of proprietary antibodies on multiple surfaces and monitored their performance under various reaction conditions. For the first time, highly specific blood grouping has been achieved on a planar microarray using directly labelled erythrocytes or a secondary labelled reagent using fluorescent signal end point readout. We have also complemented microarray data with a label-free, surface plasmon resonance-based Biacore platform data and used the real time quantitative measurement to rank anti-A antibodies according to the strength of reaction with the immobilized synthetic blood group antigen A.

Blood Grouping and Crossmatching↗
PubMed1999

Repartnering: the relevance of parenthood and gender to cohabitation and remarriage among the formerly married.

This paper is an exploratory analysis of the impact of current and anticipated parenthood on cohabitation and remarriage among those formerly living in marriage-type relationships. The focus on children is embedded within a broader analysis of repartnering which takes account of other factors, including gender. Quantitative and qualitative analyses are used, with a multivariate analysis of repartnering patterns, using data from the General Household Survey, being complemented by in-depth interview data examining the attitudes of the formerly married to future relationships. The paper demonstrates that parenthood has a statistically significant effect on the likelihood of formerly married women repartnering, with a higher number of children being associated with a lower probability of repartnering. The presence of children can work against repartnering in a variety of ways. Children place demands on their parents and can deter or object to potential partners. Parents may see their parental role as more important than, and a barrier to, new relationships. However, mothers are typically looking for partners for themselves rather than fathers for their children. Among formerly married people without children, the desire to become a parent encourages repartnering. The paper concludes that parenthood should be a key consideration in analyses of repartnering.

Adolescent↗
PubMed1981

Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.

Animals↗
PubMed1994

New non-invasive rapid diagnosis of herpes simplex virus encephalitis by quantitative detection of intrathecal antigen with a chemiluminescence assay.

A technique for detecting herpes simplex virus (HSV) antigen in CSF by measuring the antigen-antibody complex-activated complement-stimulated chemiluminescence in normal granulocytes was applied to 17 CSF samples from 10 patients with herpes simplex virus encephalitis (HSVE), and 26 samples from 22 patients with a non-HSVE brain illness. All CSF samples taken from the 5th to the 38th day (including seven from the 5th to the 10th day) of HSVE were positive, whereas those for all non-HSVE samples were negative. This assay required only five hours to yield a result.

Adolescent↗
PubMed1978

Further studies of a humoral chemotactic abnormality in glomerulonephritis.

Chemotactic activity in the sera of patients with glomerulonephritis was compared under three simultaneously performed conditions: (1) incubation with buffer at 37 degrees C (CF-UNACT); (2) incubation with immune complexes at 37 degrees C (CF-ACT); (3) immediate heating at 56 degrees C (CF-56 degrees C). In all cases the generation of chemotactic factors was terminated by standard 'heat-inactivation' at 56 degrees C. Patients' CF-UNACT was similar to that of controls; patients' CF-ACT was significantly less than controls', but patients' CF-ACT and CF-56 degrees C was significantly greater than controls'. Patients CF-ACT and CF-56 degrees C were largely C5-dependent and were quantitatively similar. These divergent abnormalities could not be explained by spontaneous in vivo or in vitro (i.e., blood clotting) generation of complement chemotactic factors, the absence of Hageman factor-dependent chemotactic activity, or the presence of humoral inhibitors in patients' sera. It appears that inital 56 degrees C heating liberates C5-dependent chemotactic activity, a procedure that is usually believed to block its formation. Terminal 56 degrees C heating after 37 degrees C incubation did not generate such activity in CF-UNACT. The duration or sequence of heating at 56 degrees C, or both, are critical determinants for final expression of chemotactic activity in patients' sera, when viewed in relation to 37 degrees C incubation with immune complexes or buffer.

Chemotaxis, Leukocyte↗
PubMed1992

Discordant secretion of relaxin by individual porcine large luteal cells: quantitative analysis by a reverse haemolytic plaque assay.

Individual large luteal cells (LLC) derived from pregnant swine differ conspicuously in their ability to secrete the peptide hormone relaxin under basal and stimulated conditions--the phenomenon of functional heterogeneity. The purpose of this study was to quantitate knowledge of this phenomenon through use of a reverse haemolytic plaque assay, a technique that utilizes antibody-directed, complement-mediated erythrocyte lysis to detect hormone secretion by single LLCs in culture. Measurement of individual plaque areas (an index of the amount of relaxin secreted) demonstrated an approximate 100-fold range in the amount of relaxin secreted by a single cell under basal conditions. This range was doubled by exposure to the phorbol ester, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA; 50 nmol/l). Under basal conditions, 50 and 80% of the total amount of relaxin was secreted by approximately 10 and 30% of all LLCs respectively. The size of these fractions was not influenced by the time of incubation (1-8 h), or by the presence of either of two non-specific stimulatory relaxin secretagogues, PMA (50 nmol/l) or arachidonic acid (1 mumol/l). The unimodal frequency distribution of plaque areas (under basal or stimulated conditions) suggests that relaxin-secreting LLCs comprise a discrete functional population of secretory cells, at least under these experimental conditions. We conclude that a remarkably small fraction of LLCs secretes the majority of relaxin, and that the size of this fraction was not influenced by time or secretagogues.

Animals↗
PubMed2003

Pathogenetic aspects of immune deficiency associated with beta-thalassemia.

Infectious complications constitute the second most common cause of mortality and a main cause of morbidity in beta-thalassemia. Besides the high risk of blood-borne infections associated with multiple transfusions, the increased susceptibility of these patients to infectious diseases has been attributed to a coexistent immune deficiency. Immune abnormalities have also been held responsible for the frequent occurrence of malignancies in beta-thalassemia, especially leukemia and lymphomas. Recent studies on immune competence in beta-thalassemia have revealed numerous quantitative and functional defects, involving T and B lymphocytes, immunoglobulin production, neutrophils and macrophages, chemotaxis, and phagocytosis, as well as the complement system. Regarding pathogenesis, iron overload, a primary complication of both thalassemia itself and transfusion therapy, is thought to be the main precipitating mechanism, due to the important immunoregulatory properties of iron and its binding proteins; iron excess may derange the immune balance in favor of the growth of infectious organisms. Other factors include multiple transfusions, associated with constant allo-antigenic stimulation, as well as with transmission of immunosuppressive viruses; splenectomy, resulting in increased susceptibility to infections by encapsulated bacteria and to immune system modifications; low levels of zinc, another immune regulator; iron chelation therapy, which predisposes to serious infections by yersinia species; and the circulation of abnormal native thalassemic erythrocytes, forming another permanent immune stimulus. Thus surveillance for infections in patients with beta-thalassemia is crucial, while further studies are warranted on immune function abnormalities and the implicated mechanisms.

Humans↗
PubMed1987

In vivo immunosuppression by pan-T cell antibodies relates to their isotype and to their C1q uptake.

There is considerable interest in the use of monoclonal anti-T cell antibodies for immunosuppression during organ transplantation. However, the in vitro cytotoxic titers of these monoclonal reagents do not correlate with their immunosuppressive potency when injected in vivo. A relationship nevertheless seems to exist between immunosuppression and the isotype of anti-mouse Thy-1 antibodies, because among several anti-Thy-1 antibodies of mouse and rat origin, the only two found to cause immunosuppression in vivo belonged to the rat IgG2b and mouse IgG2a isotype. We show here that a quantitative positive correlation exists between an antibody-induced humoral effector mechanism and immunosuppression. We measured the uptake of the C1q complement subunit by polyclonal rabbit and rat anti-thymocyte globulin and also seven monoclonal anti-Thy-1 antibodies in an immunohistochemical assay or a radioimmunoassay. Immunosuppression was studied in a murine graft-vs-host and skin allograft model. Our results suggest strongly that a stable association between the C1 protein and a potential binding antibody is an essential prerequisite of antibody-dependent cell inhibition in vivo that suppresses the immunoresponse against strongly incompatible transplantation antigens.

Animals↗
PubMed1994

HRRI: a high resolution radioimager for fast, direct quantification in in situ hybridization experiments.

We present a high-speed, high-resolution beta imager. It has been developed to be used in in situ hybridization experiments, either instead of or in complement with autoradiographic film and emulsions that are currently used for these experiments. It allows the user to locate and perform quantitative analyses of (3H-, 14C-, 35S-, 32P-, 125I-) labeled molecules with a 15-microns spatial resolution on a 1.2 cm2 area. We have combined recent techniques (specific scintillator thin sheets and intensified charge-coupled device [CCD]) so that this imager offers a wide dynamic range and real-time acquisition. Several biological applications will be discussed.

Animals↗
PubMed1993

Glomerular C3c localization indicates ongoing immune deposit formation and complement activation in experimental glomerulonephritis.

In antibody-mediated glomerular disease, deposits of C3 (C3b) are common and are degraded by factor I to C3c and C3d. However, the kinetics of C3b degradation in glomerulonephritis have not been defined. To do this, we studied three models of complement-dependent glomerulonephritis with established C3 deposits (passive Heymann nephritis, cationized immunoglobulin G membranous nephropathy, and concanavalin A-anticoncanavalin A glomerulonephritis). C3b deposition was halted by administration of cobra venom factor, and the disappearance of C3c and C3d from glomeruli was measured with specific antibodies and quantitative fluorescence densitometry. Results showed that C3c deposits were reduced by over 85% within 24 hours in all three models. C3c clearance was unaffected by site or mechanism of deposit formation. C3d deposits persisted despite lack of ongoing complement activation. In passive Heymann nephritis when disease activity was monitored by urinary C5b-9 excretion, C3c was cleared in parallel with return of urine C5b-9 excretion to normal values. We conclude that glomerular deposits of C3c are cleared within 24 hours of cessation of complement activation. Positive staining for C3 utilizing antibody specific for the C3c portion documents recent complement activation usually reflecting new immune deposit formation.

Animals↗
PubMed1991

Effect of cell-bound proteins on the in vivo survival of circulating blood cells.

Normal circulating red blood cells (RBCs) and platelets have been shown to have small amounts of IgG on their membranes. The cell-bound IgG may be cytophilic (IgG nonspecifically adsorbed from the plasma) and/or IgG autoantibody. It has been suggested that most of the RBC-bound IgG is on older RBCs and is an autoantibody directed against senescent cell antigen (SCA). The accumulation of this RBC-bound IgG leads to Fc-dependent removal of senescent RBCs by macrophages in the reticuloendothelial system. RBCs also have complement components on their membrane; it is not clear how this accumulates on RBCs and whether it has a physiologic function. This small amount of nonpathogenic RBC-bound IgG is not detected by the antiglobulin test. It is still unclear whether the major difference between pathogenic and nonpathogenic IgG autoantibodies is qualitative, quantitative, or both. Seemingly healthy blood donors (1 in 1,000 donors) and patients without hemolytic anemia (up to 8% of patients) have increased amounts (greater than 200 molecules/RBC) of RBC-bound IgG and complement that is detected by the antiglobulin test. This RBC-bound IgG has been shown to be an IgG autoantibody directed against blood group antigens, and/or IgG anti-idiotype, and/or IgG nonspecifically adsorbed onto the RBC membrane when plasma IgG levels are high. Most patients with autoimmune hemolytic anemia (AIHA) have RBC-bound IgG and/or complement detectable by the antiglobulin test. Most of the RBC-bound IgG is of the IgG1 subclass, whether one examines the RBCs of healthy blood donors or hospitalized patients with and without AIHA. Although the quantity of RBC-bound IgG is generally higher in patients with AIHA, there is no clear correlation with the quantity of RBC-bound IgG and the rate of in vivo RBC destruction. There is some recent evidence that the SCA autoantibody may at times be pathogenic and cause autoimmune disease.

Anemia, Hemolytic↗
PubMed1998

In vivo recruitment of neutrophils: consistent requirements for L-arginine and variable requirements for complement and adhesion molecules.

The current studies examined the mechanisms of neutrophil recruitment into the rat peritoneal cavity following injection of glycogen and into rat lungs following alveolar deposition of IgA immune complexes or airway instillation of phorbol ester (PMA). Unexpectedly, in each model a requirement for L-arginine for neutrophil recruitment was demonstrated, since administration of the L-arginine analogue, NG-monomethyl L-arginine acetate (L-NMA), greatly reduced neutrophil accumulation as assessed by quantitation of neutrophils in peritoneal exudates and bronchoalveolar lavage fluids, and by lung myeloperoxidase content. In the case of IgA immune complex deposition, lung recruitment of neutrophils was also suppressed by soluble recombinant human complement receptor-1 (sCR1) and antibody to CD18 but not by antibody to E-selection. In contrast, neutrophil accumulation following airway instillation of PMA exhibited, surprisingly, no requirement for complement but requirements for both E-selection and CD18. These data demonstrate variable requirements for complement, E-selectin and CD18 but a consistent requirement for L-arginine for neutrophil recruitment. These findings provide evidence suggesting that L-arginine or its derivatives regulate neutrophil recruitment.

Alveolitis, Extrinsic Allergic↗
PubMed1995

Quantitative and qualitative aspects of spontaneous specific-locus mutation in the ad-3 region of heterokaryon 12 of Neurospora crassa.

The data from forward-mutation experiments to obtain specific-locus mutations at 2 closely linked loci in the adenine-3 (ad-3) region of heterokaryon 12 (H-12) of Neurospora crassa have been tabulated to determine the frequency of spontaneous ad-3 mutations and to determine the percentages resulting from each of the 2 major genotypic classes: gene/point mutations and multilocus deletion mutations. Gene/point mutations at the ad-3B locus (ad-3BR) have been characterized to determine the percentage showing allelic complementation to obtain a presumptive identification of the genetic alteration in each mutation at the molecular level. Data from experiments performed at 2 different laboratories have been compared to assess the interlaboratory reproducibility of quantitative data on H-12. No difference was found between the frequencies of spontaneous specific-locus mutations in the ad-3 region. Genetic analysis of 172 ad-3 mutants demonstrated that specific-locus mutations in the ad-3 region result from both gene/point mutations (82.0% [141/172]), and multilocus deletion mutations (14.5% [25/172]). Heterokaryon tests for allelic complementation demonstrated that 52.5% (53/101) spontaneous ad-3BR mutants show allelic complementation, and result from single base-pair alterations. In addition, 100% (25/25) of the spontaneous multilocus deletion mutations result from the 3 smallest sized genotypic subclasses. The implications of the present experimental data for the validation of the ad-3 specific-locus assay system in Neurospora are discussed.

Alleles↗
PubMed1977

Intra-individual variation in the concentrations of IgA, IgG, IgM, and complement component C3 in serum of a normal adult population.

A recent study [Clin. Chem. 22, 1635 (1976)] reported intra-individual variation in 10 serum proteins to be much smaller than interindividual variation. We report results of a similar study involving about 700 apparently healthy adults in whom we estimated the relative magnitude of the intra- and interindividual variation in serum IgA, IgG, IgM and complement component C3. Specimens were collected from each subject weekly for as long as 10 weeks (average, four weeks). The four serum proteins were quantitated by radial immunodiffusion by the maximal-diffusion technique. Traditional 95% reference intervals were computed relative to WHO reference preparations for the immunoglobulins. For C3, the reference interval was computed relative to a commercial reference preparation. We, too, found the ratios of intra-individual to interindividual variation for adults to be so small that the traditional reference intervals do not have the assumed diagnostic sensitivities. Furthermore, these ratios did not change after dividing the study population into subgroups according to sex and age; evidently such subgrouping do not improve the diagnostic sensitivity. The relatively small intra-individual variations were also observed at the extremes of protein concentration ranges.

Adolescent↗