bluuebunny/crossref_metadata_2025_split huggingface dataset:
Demonstration and quantitation of activation of the first complement in human serum
Authors: Ziccardi RJ , Cooper NR 
Published: June 2004

Activation of the first component of human complement (C1) in human sera can be readily detected in double immunodiffusion studies with anti-C1q, anti- C1r, and anti-C1s as it produces a characteristic pattern quite different from that of precursor C1. Native macromolecular C1 gives a continuous line of precipitation with antisera to C1q, C1r, and C1s in double diffusion studies. After activation of C1 by incubation of serum with complement activators, three major changes occurred in the Ouchterlony pattern. First, spurring of the C1s precipitin line over that of macromolecular C1, indicating release of C1s from C1, was observed with low doses of activator. Release of C1s was quantitated by single radial diffusion and shown to be complete with the highest activator dose examined. Second, C1q was released with larger activator doses as shown also by spurring of the precipitin line due to this component over the remaining macromolecular C1. Third, and most surprising, C1r antigenicity was progressively lost as the activator dose was increased and no C1r line remained with the highest dose of activator tested. This was not true with C1s as there was no change in the total C1s concentration in serum incubated with various activator doses.
These observations provide two approaches to the quantitation of C1 activation in human serum. First, C1r and C1s can be quantitated by single radial diffusion. A decrease in the C1r:C1s ratio correlates with activation. Second, C1s released by the activation can be quantitated by single radial diffusion if the agarose contains high concentrations of anti-C1q to confine C1, also containing C1s, to the area near the application well, and lesser concentrations of anti-C1s to permit free C1s to produce a measurable ring. The extent of release of C1s also correlates with activation.
These immunochemical techniques to quantitate C1 activation directly inserum do not require specialized reagents. It is hoped that they will be useful in screening pathological sera and in monitoring the status of the complement system in patients.
Presence of anti-Sm reactivity in autoimmune mouse strains
Authors: Eisenberg RA , Tan EM , Dixon FJ 
Published: June 2004

The investigation of the fine specificities of antinuclear antibodies (ANAs) has been fruitful in terms of the nosology and immunopathogenesis of human autoimmune syndromes. Particular reactivities serve as markers, in that patients with certain syndromes have a much higher incidence of such ANAs than do patients with other diseases. In this category is the almost exclusive against the nuclear acidic protein Sm. Reactivity to Sm can be detected by precipitation in agar, complement fixation, or passive hemagglutination (1,2).
Autoimmune mouse strains have also provided a fertile field for the investigation of the basic phenomena of self-activity. In particular, the NZB strain and its hybrid NZB x NZW have been considered excellent models for human SLE and have therefore been studied in great detail (3,4).
In addition, Murphy et al at The Jackson Laboratory, Bar Harbor, Maine, have developed several new inbred mouse strains that spontaneously develop SLE-like syndromes (5,6). These are the BXSB strain, which has a male dominant disease characterized by little antiative DNA antibody; the MRL/1, which develops massive, nonmalignant lymphadenopathy, associated with enormous increases in serum immunoglobulin levels and fulminant renal disease; and the MRL/n, which does not develop SLE-like disease until well into the 2nd yr of life, but like the MRL/1 develops high titers of ANA and fatal glomerulonephritis. The MRL/1 differs from MRL/n in only about 10 percent of its genome, including the gene responsible for the MRL/1's lymphoproliferation.
In the current study, we have used the technique of double immunodiffusion (ID) in agarose with standard human reference sera (of known ANA specificity) to survey a large number of mice from the NZB x NZW, MRL/1, MRL/n, BXSB, and other strains. We report here the finding of the anti-Sm marker antibody almost uniquely in MRL/1 and MRL/n animals. These two related strains may serve as experimental models to explore the mechanism stimulating the production of this unique autoantibody in SLE.
Human C4-binding protein. I. Isolation and characterization
Authors: Scharfstein J , Ferreira A , Gigli I , Nussenzweig V 
Published: June 2004

C4-binding protein (C4-bp), a new component of the complement system, was isolated from human plasma by precipitation with polyethyleneglycol, followed by chromatography on ion exchangers. C4-bp was identified on sodium dodecyl- sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by two independent criteria: its ability to bind to C4b, and immunoprecipitation with a monospecificantiserum.
Purified C4-bp is a 10.7 s glycoprotein. It consists of several disulfide bonded subunits of mol wt 70,000 daltons. Under nonreducing conditions, its mol wt has been estimated on SDS-PAGE as 540- 590,000 daltons.
C4-bp moves as a slow B-globulin at pH 8.6 in the absence of free divalent cations, but when the buffers contain Ca(++)-lactate, C4-bp is a gamma globulin.
Purified C4-bp binds to purified C4b. The reaction proceeds in the presence or absence of divalent cations and is not inhibited by diisopropylfluorophosphate. The C4b/C4-bp complexes have sedimentation coefficients between 15 and 17 s on sucrose gradient ultracentrifugation, and can be readily identified by crossed immunoelectrophoresis (CIE). The complexes move faster toward the anode than either protein.
C4-bp is multivalent. Saturation is reached at molecular ratios of C4b/C4- bp of between 4 and 5.
The interaction between C4b and C4-bp may complicate the electrophoretic patterns of these proteins in normal human serum, if the complement system is activated before or during the run. However, in EDTA-plasma, native C4 and C4-bp do not form stable complexes and can be identified in separate peaks after CIE.
In vitro maturation of immature thymocytes into immunocompetent T cells in the absence of direct thymic influence
Authors: Irle C , Piguet P-F , Vassalli P 
Published: June 2004

Peanut lectin (PNL) binds to a majority of mouse thymocytes (Thc) in suspension. By using cell affinity chromatography on a column of anti-PNL antibody, Thc populations at least 96 percent pure in PNL + or - cells, as judged by immunofluorescence, were obtained. PNL(+) cells are rich in Thy 1 and poor in H(2) antigens, cortisone sensitive, unresponsive to phytohemagglutinin (PHA), and immunologically incompetent, as judged by mixed lymphocyte reaction, popliteal lymph node graft-versus-host assay, and by testing helper activity in a primary in vitro antibody response to sheep erythrocytes; the converse is true of PNL(-) cells. Thus, PNL(+) and (-) cells appear to correspond to cortical and medullary Thc, respectively, as previously suggested. In culture, PNL(+) Thc show poor viability and a weak proliferative response to concanavalin A (Con A), except when supernate (SUP) of 24 h Con A stimulated lymph node lymphocyte cultures, or irradiated lymph node cells, are added, in which cases a strong proliferative response to the mitogen is observed. A variety of control experiments showed that the proliferating cells did not result from preferential stimulation of a few contaminating PNL(-) Thc present in the PNL(+) Thc cultures. The blasts resulting from PNL(+) Thc proliferation display mitogen-induced cytotoxicity, and give rise to a population of medium-sized lymphocytes, mostly PNL(-), poor in Thy 1 and rich in H(2) antigens, PHA responsive, and immunologically competent in the above-mentioned assays.
Fresh PNL(+) Thc responded in mixed lymphocyte reaction in the presence of SUP (lectin depleted) and since incubation in SUP alone did not confer reactivity on PNL(+) Thc, it appears therefore that (a) immature Thc possess alloantigen and mitogen-specific surface receptors but lack the capacity to respond by proliferation to receptor triggering without the help of extracellular factor(s) released by mature lymphoid cells stimulated by mitogens (b) cell division is associated with the acquisition of immunological responsiveness, characteristic of mature T lymphocytes. The implications of these findings for the ontogenesis of thymus-derived lymphocytes, and for the possible traffic of Thc within and from the thymus, are discussed.
H-2 antigens of the thymus determinelymphocyte specificity
Authors: Fink PJ , Bevan MJ 
Published: June 2004

After immunization, normal H-2 heterozygous mice (for example H-2(b) × H-2(d)) generate two populations of cytotoxic effector T cells, one specific for target cells expressing H-2(b)-plus-antigen and the other specific for H- 2(d)-plus-antigen. With a multideterminant antigen, these two populations have about the same activity. We show here that the H-2 type of resident cells in the thymus determines the H-2 preference of cytotoxic T lymphocytes. F(1)(B 10 × B 10.D2) (H-2(b) × H-2 (d)) mice were thymectomized, lethally irradiated, and reconstituted with T-cell-depleted syngeneic hematopoietic cells. Groups of such ATXBM mice were grafted subcutaneously with neonatal thymus lobes from parental mice, either B10 (H-2 (b)) or B10.D2 (H-2(d)). 2-3 mo later, the mice were immunized against the minor histocompatibility antigens on F(1)(BALB/c × BALB.B) cells and assayed for cytotoxic T-cell activity. H-2(b) × H-2(d) ATXBM mice with H-2(b) thymus grafts responded to antigen-plus-H-2(b) much better than to antigen-plus-H-2(d), and vice versa for the mice with H-2(d) thymus grafts. As judged by antiserum treatment, the effector cells were of F(1) origin.
To explore the possibility that the "thymus preference" may have been due to suppression of T-cell activity, nonimmune spleen and lymph node cells from normal H-2(b) × H-2(d) mice and cells from H-2(b) × H-2(d) mice bearing a homozygous thymus were mixed 1:1 and immunized in adoptive transfer. The mixture responded to antigen-plus-H-2(b) and antigen-plus-H-2(d) equally well, demonstrating that the cells that showed a "thymus preference" could not suppress a response to antigen in association with the nonthymic H-2 type. We conclude from these and other experiments that H-2 antigens present on resident cells of the thymus determine the spectrum of specificity of T cells which mature in that thymus and eventually make up the peripheral T- cell pool.
Mechanisms of clonal abortion tolerogenesis. I. Response of immature hapten- specific B lymphocytes
Authors: Nossal GJV , Pike BL 
Published: June 2004

B lymphocytes with receptors specific for the hapten fluorescein (FLU) were prepared from the spleens of mice of various ages. For most experiments, a one-step fractionation procedure based on the adherence of FLU-specific cells to FLU-gelatin was used. For some experiments, a subset of higher FLU-binding capacity was prepared from the FLU-gelatin binding population through the use of the fluorescence-activated cell sorter (FACS). FLU-specific B cells were placed into microculture with either FLU(3.6)-human gamma globulin (FLU(3.6)HGG) or FLU(12)HGG usually for 24 h at 37 degrees C. The tolerogen was then removed and 0.1 μg/ml of a T-independent antigen, FLU-polymerized flagellin, was substituted. 3 days later, cells were harvested from the microcultures and assayed for FLU-specific plaque-forming cells to determine any reduction in clonable hapten-specific B cells which the tolerogenesis treatment might have induced.
The results showed that with FLU(3.6)HGG, hapten-specific newborn B cells could be tolerized at 1,000-fold lower tolerogen concentrations than adult splenic B cells of equal antigen-binding capacity. The high-avidity subset was even more susceptible to tolerance induction. Tolerance could be induced within 8 but not within 2 h, and at lower tolerogen concentrations, longer periods of tolerogenesis were required for a given effect. Using a 24-h tolerogenesis phase, 50 percent reduction in clone frequency among newborn FLU-gelatin fractionated cells was achieved at 0.08 μg/ml of FLU(3.6)HGG. Tolerance induction in immature B cells was inhibited by the concomitant presence of a polyclonal B-cell activator, Escherichia coli lipopolysaccharide (LPS) but tolerance once induced, was stable to challenge with LPS. Tolerogenesis was hapten specific. The proportion of tolerizable cells in spleens decreased with increasing age, reaching 50 percent at around 9 days.
FLUI(12)HGG proved a more powerful tolerogen than FLU(3.6)HGG. It had an effect on adult cells, 50 percent reduction in clone frequency being noted at around 1 μg/ml. However, and in contrast to results claimed for other T- independent systems, there still was a major difference between immature and mature B cells, the immature cells displaying much greater sensitivity to tolerogenesis.
Role of phagocytosis in the activation of macrophages
Authors: Schnyder J , Baggiolini M 
Published: June 2004

Macrophages were obtained by peritoneal lavage from untreated mice or from mice which had received either Brewer's thioglycollate broth or a suspension of streptococcus A cell walls intraperitoneally 4 days before. 3 h after harvesting, adherent cells from untreated mice were allowed to phagocytose zymosan, formaldehyde-treated sheep erythrocytes, or latex beads. Phagocytosis was stopped after 1 h and culture was continued for up to 10 days. Phagocytosis of zymosan or sheep erythrocytes triggered the immediate release of lysosomal glycosidases, stimulated the synthesis of cellular lactate dehydrogenase, and induced the delayed production and secretion of plasminogen activator . No such changes were observed upon phagocytosis of latex. Although all three particles used were phagocytosed, only zymosan and sheep erythrocytes stimulated glucose oxidation via the hexose monophosphate shunt.
Similar findings were obtained in macrophages elicited with streptococcus A cell walls after zymosan phagocytosis. Thioglycollate-elicited macrophages, however, which were already secreting lysosomal hydrolases and plasminogen activator, could not be activated further by zymosan.
The results of this study show that macrophages become activated after phagocytosis of particles that stimulate the activity of their hexose monophosphate shunt. The triggering event appears to be the burst of shunt activity itself or shunt-related biochemical reactions rather than phagocytic uptake per se or particle-dependent complement activation by the alternative pathway. Once initiated, macrophage activation proceeds independently of the intracellular fate of the ingested material .
Monoclonal origin of B lymphocyte colony-forming cells in spleen colonies formed by multipotential hemopoietic stem cells
Authors: Lala PK , Johnson GR 
Published: June 2004

Spleen colonies produced by transplanting lethally irradiated mice with either 12 day fetal liver or adult bone marrow cells were found to contain B- lymphocyte colony-forming cells (BL-CFC) . The proportion of BL-CFC positive spleen colonies did not increase substantially between 8 and 14 days after transplantation, the range being 18-45 percent. However, the absolute number of BL-CFC per spleen colony varied considerably (between 1 and 10,318), although the majority of colonies contained less than 200 BL-CFC. Irrespective of the time after transplantation, smaller spleen colonies were found to have a higher frequency of BL-CFC than larger spleen colonies.
To determine the possible clonal origin of BL-CFC from spleen colony- forming unit (CFU-S), CBA mice were injected with equal numbers of CBA and CBA T(6)/T(6) fetal liver or adult bone marrow cells. Analysis of 7-15-day spleen colonies demonstrated that 90 percent were either exclusively T(6) positive or T(6) negative and approximately equal numbers ofboth colony types were observed. B-lymphocyte colonies were grown and successfully karyotyped from 19 spleen colonies. When compared with the original spleen colony karyotype the B-lymphocyte colony cells karyotype was identical in all 19 cases. In 3 of the 19 colonies analyzed a mixture of T(6) positive and T(6) negative karyotypes was present and identical proportions of the karyotypes were present in the pooled B-lymphocyte colony cells and spleen colony cells.
The data indicate that the B-lymphocyte colony-forming cells detected in spleen colonies are genuine members of the hemopoietic clone derived from the initiating hemopoietic stem cell (CFU-S).
Secretion of plasminogen activator by bone marrow-derived mononuclear phagocytes and its enhancement by colony-stimulating factor
Authors: Lin H-S , Gordon S 
Published: June 2004

We have studied the production of plasminogen activator (PA) by mononuclear phagocytes derived from mouse bone marrow precursor cells (CFU-C) in culture. Bone marrow-derived macrophages (BMDM) obtained after 6-8-d cultivation in a liquid medium containing L-cell-conditioned medium (LCM), a source of colony stimulating factor (CSF), showed a high level of fibrinolytic activity comparable to that of thioglycollate medium-induced peritoneal macrophages (TPM) and at least 20-fold higher than that of resident peritoneal macrophages (RPM). Fibrinolysis was a result of active secretion of PA into the culture medium and plaques of caseinolysis could be detected by an overlay assay over all macrophage colonies formed after cloning of bone marrow cells in culture.
When the fibrinolytic activity of BMDM harvested at different times was investigated, it was found that the level of PA activity of a given BMDM population correlated well with the incidence of cells (5-15 percent) able to proliferate and form colonies in agar after 7-14 d, somewhat more slowly than CFU-C. This correlation between the level of PA secretion and the incidence of agar colony-forming cells was also found with other mononuclear phagocyte populations. Active fibrinolysis and slow growing colony-forming cells were observed at the same time as adherent macrophages appeared, 2-3 d after the start of bone marrow culture, they persisted for 10 d before declining.
Some of the factors which influenced PA production by BMDM were examined. Fibrinolysis could be enhanced two- to fourfold by exposing the cells for 4 h to concanavalin A (Con A), to medium conditioned by Con A-stimulated spleen cells and to LCM, but not by phagocytosis of latex particles. The substance in LCM that stimulated PA production appeared to be identical to CSF. Mononuclear phagocyte targets differed in their response to LCM, which stimulated fibrinolysis readily in BMDM, to a lesser extent in TPM and not at all in RPM.
We conclude that CSF stimulates both proliferation and fibrinolytic activity in BMDM and that the level of macrophage activation, as defined by PA production, can be further enhanced by lymphokines. Induction of PA in BMDM provides a rapid and sensitive assay for measuring the activity of CSF and defining its role in macrophage activation.
Humoral and cell-mediated immune responses in fully allogeneic bone marrow chimera in mice
Authors: Onoe K , Fernandes G , Good RA 
Published: June 2004

AKR mice were protected from lethal irradiation and established as long-lived chimeras by transplanting allogeneic C57BL/6 (B6) bone marrow that had been treated in vitro with anti-Thy-1 antiserum without complement. In these chimeras, which were designated [B6 {arrow} AKR], virtually all the thymus and spleen cells were shown to be derived from the B6 donor; several immune functions studied in these chimeras were as follows: (a) The chimeric mice were tolerant of histocompatibility antigens of both donor and recipient strain and nearly fully reactive to antigens of third party, as revealed by Simonsen's splenomegaly assay. The tolerance of these chimeras could not be attributed to suppressor cells but was compatible with clonal depletion. (b) Proliferative responses to concanavalin A, phytohemagglutinin, and lipopolysaccharide as well as natural killer and antibody-dependent cell- mediated cytotoxicity activity of the chimeric mice was normal. (c) Plaque- forming cell (PFC) assays of antibody responses to sheep erythrocytes (SRBC) showed gross deficiency in the primary response of the [B6 {arrow} AKR] and [AKR {arrow} B6] chimeras.
By contrast, [B6-H-2(k)(E(k)) {arrow} AKR] H-2-compatible chimeras and [AKR {arrow} AKR] syngeneic marrow transplanted mice had normal primary PFC responses. PFC responses after secondary stimulation with SRBC, however, revealed vigorous direct plaque formation and substantial but somewhat smaller indirect plaque formation in the [B6 {arrow} AKR] chimeras. This observation favors operationally the concept of adaptive differentiation proposed by Katz et al. (44). (d) Analysis of ability of the chimeras to develop and express delayed-type hypersensitivity responses to contact sensitizer (2,4-dinitro-l-fluorobenzene [DNFB]) showed no apparent immunodeficiency of either chimeras to this form of immunization. Development of immunologic tolerance to DNFB, however, was grossly deficient in [B6 {arrow} AKR] chimeras but normal in [AKR {arrow} AKR], [B6 {arrow} B6], and [E(k) {arrow} AKR] chimeras.
These findings indicate that full chimeras across major histocompatibility complex have considerable immunologic vigor even though primary immune responses that require histocompatibility between interacting cell types are initially defective.
Lyb-2 system of mouse B cells. Evidence for a role in the generation of antibody-forming cells
Authors: Yakura H , Shen F-W , Kaemmer M , Boyse EA 
Published: June 2004

The Lyb-2 cell-surface alloantigens of the mouse are selectively and perhaps exclusively expressed in the B lymphocyte lineage, but not on antibody- forming cells. Thus if the Lyb-2 molecule is concerned in specific B cell function, it must participate in the generative phase of the antibody response.
Accordingly, monoclonal Lyb-2 antibody was found to depress the plaque- forming cell (PFC) response to sheep erythrocytes in 5-d Mishell-Dutton assays when added within the first 3 d of culture, but not later. The rate of PFC generation was not affected, signifying an absolute reduction in the number of PFC generated. Because reduction of PFC counts by Lyb-2 antibody was not affected by exclusion of Lyt-2(+) T cells, it is unlikely that the reduction depends on augmented suppression by T cells. Augmented B cell- mediated suppression is also unlikely, because the PFC response of serial combinations of congenic Lyb-2.1 and Lyb-2.2 cells, in the presence of monoclonal Lyb-2.1 antibody, was reduced only in direct proportion to the number of Lyb-2.1 cells present. The PFC response of Lyb-2.1/Lyb-2.2 heterozygous cells was not reduced by Lyb-2.1 antibody, presumably because generation of PFC is impeded only if most Lyb-2 sites are blocked. Further evidence that the molecule identified by Lyb-2 plays a critical role in the generation of antibody-forming cells (AFC) in response to T-dependent antigen comes from the finding that Lyb-2 antibody does not reduce the PFC response to the T-independent antigens trinitrophenylated (TNP) Brucella abortus and TNP-FicolI, although elimination of Lyb-2(+) cells from the starting population by Lyb-2 antibody and complement reduces the PFC response to T- dependent and T-independent antigens alike.