bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of the Capsular Polysaccharide in the Activation of the Alternative Pathway by the Pneumococcus
Authors: Winkelstein J. A. , Bocchini J. A. , Schiffman G.
Published: January 2023

The alternative pathway plays a significant role in the non-immune host's defense against the pneumococcus. The following studies concern the role of the capsular polysaccharide in the activation of the alternative pathway by the pneumococcus.

Some pneumococcal capsular polysaccharides, but not all, are able to activate the alternative pathway. Soluble purified capsular polysaccharide types 1, 4, and 25 activate C3 in C4-deficient guinea pig serum (C4D GPS), where types 2, 3, 14, and 19 do not. Since capsular polysaccharides exist in their native form attached to the pneumococcal surface, selected capsular polysaccharides were also tested when coupled by CrCl3 to a particulate carrier, sheep erythrocytes. When attached to sheep erythrocytes, capsular polysaccharide types 2 and 3 failed to consume C3 in C4D GPS, paralleling the results obtained when they were in solution. In contrast, type 25 capsular polysaccharide coupled to erythrocytes not only consumed C3 in C4D GPS, as it had when in solution, but also initiated alternative pathway lysis of the erythrocytes.
Pre-B Cells: Bone Marrow Persistence in Anti-µ-Suppressed Mice, Conversion to B Lymphocytes, and Recovery after Destruction by Cyclophosphamide
Authors: Burrows Peter D. , Kearney John F. , Lawton Alexander R. , Cooper Max D.
Published: January 2023

Chronic treatment of mice from birth with anti-µ antibodies aborts development of B lymphocytes and plasma cells. In these studies we show that bone marrow from anti-µ-treated mice contains a population of cells with cytoplasmic IgM, but which lack detectable cell-surface IgM. These cells are analogous to pre-B cells, defined in ontogenetic studies as the immediate precursors of B lymphocytes. Pre-B cells from bone marrow of anti-µ treated mice retain their functional integrity, as evidenced by their ability to give rise to sIgM+, LPS-responsive lymphocytes in culture. We also show that cyclophosphamide treatment destroys pre-B cells and that recovery of pre-B cells in bone marrow precedes the regeneration of sIgM+ B lymphocytes. Generation of B lymphocytes in adult mice apparently occurs exclusively in the bone marrow because induction of extramedullary hemopoiesis in spleen was not accompanied by the appearance of pre-B cells in that organ.
Inhibition of Cytolytic T Lymphocyte Activity with Subcellular Alloantigen Preparations and with Unlabeled Allogeneic Target Cells
Authors: Linna T. Juhani , Engers Howard D. , Cerottini Jean-Charles , Brunner K. Theodor
Published: January 2023

Studies of the specificity of target cell lysis by cytolytic T lymphocytes (CTL) are usually based on assays measuring the competitive inhibition of radioactively labeled target cell lysis with intact unlabeled cells carrying antigens of the same or different specificities. So far, attempts to inhibit lysis with subcellular antigen preparations have met with little success. In order to obtain more favorable conditions for competitive inhibition of target cell lysis with subcellular antigens, we have introduced two modifications of the techniques of CTL induction and lysis, including a) generation of CTL by stimulating spleen cells from alloimmune mice with particulate alloantigen preparations in vitro, and b) restriction of effector to target cell interaction (in the presence or absence of particulate antigen) to 15 min, thus restricting lysis to those target cells initially bound to CTL. With these modifications of the standard 51Cr-release assay, specific inhibition by unlabeled target cells was readily and consistently demonstrable. Subcellualr particulate antigen preparations were also able to inhibit specifically CTL activity, but the inhibition was usually less marked than with intact target cells, and not every antigen preparation had the capacity to inhibit in an immunologically specific way.
Tumor Cell Cytostasis by Macrophages and Antibody in Vitro
Authors: Pasternack Gary R. , Johnson Robert J. , Shin Hyun S.
Published: January 2023

Tumor cells in a cytostatic state caused by macrophages and antibody were isolated. Such suppressed cells excluded vital dye, incorporated uridine and leucine, and metabolized glucose. They did not, however, incorporate thymidine, nor did they resume cell division in culture. During prolonged culture, these cells eventually died. In this system, cytostasis was an all-or-nothing phenomenon at the level of the individual cell. Once in the cytostatic state tumor cells did not resume proliferation.
Pharmacologic Control of the Hormonally Modulated Immune Response
Authors: Pierpaoli Walter , Maestroni Georges J. M.
Published: January 2023

A recently developed pharmacologic means for suppressing acquired immunity by drugs acting on neuroendocrine regulation has been applied to transplantation immune reactions. A number of drugs have been tested singly and in combination for their capacity to suppress the immune response of mice grafted with allogeneic skin. Another model involved newborn F1 hybrid recipients inoculated with spleen cells from donors of parental strains that had been made specifically “unresponsive” by drug and alloantigen treatment. These procedures led to the identification of a combination of four drugs that induced a remarkable delay in allograft rejection and a prolonged unresponsiveness to alloantigens. This combination of drugs also abrogated the graft-vs-host-runting syndrome in newborn hybrid recipients.
Specific Suppression of Anti-Hapten Reaginic Antibody Titers with Hapten-Coated Liposomes
Authors: Schwenk Robert , Lee Weng Y. , Sehon Alec H.
Published: January 2023

Reaginic antibodies of DNP and ovalbumin (OA) were induced in B6D2F1 mice by a single i.p. injection of 1 µg of DNP3-OA suspended with 1 mg of Al(OH)3 in 0.5 ml of saline. The anti-DNP reaginic antibody titers were markedly depressed by treatment of mice with DNP-coated liposomes. This treatment, however, did not affect the level of antibody formation to OA.
Alteration of the Fatty Acid Composition of Membrane Phospholipids in Mouse Lymphoid Cells
Authors: Mandel Gail , Shimizu Stanley , Gill Ronald , Clark William
Published: January 2023

A simple method is described for introducing exogenous fatty acids into the membrane phospholipids of the murine leukemia cell EL-4, and into the membrane phospholipids of resting mouse lymphocytes. The method involves culturing of the cells with free or methylated fatty acids at concentrations up to 50 µg/ml. The presence of serum in the culture medium does not interfere with fatty acid uptake, but does increase the growth rate and viability of the cells. Membrane lipid composition returns to normal after the cells are grown in medium without exogenous fatty acid. Fractionation of the cell membranes confirmed that exogenous fatty acids were incorporated into the phospholipids of the plasma membrane.
Functional Properties of EL-4 Tumor Cells with Lipid-Altered Membranes
Authors: Mandel Gail , Clark William
Published: January 2023

The effect of fatty acid substitution of membrane phosphatides on a number of surface-mediated phenomena in EL-4 cells was examined. Tumor cells were grown in the presence of fatty acids that could be expected, on the basis of their physical properties, either to stiffen or fluidize the plasma membrane. Substitution of EL-4 cell membrane phosphatides with as much as 74% nonade-canoic acid (19:0) had no effect on either conjugation with effector cells or subsequent cytolysis by the effector cells. Substitution with linolenic acid (18:3) or elaidic acid (18:1trans) likewise affected neither conjugation nor cytolysis. Substitition with these fatty acids also had no effect on the susceptibility of EL-4 cells to cytolysis by antibody plus complement. On the other hand, the rate of patching of H-2 surface antigens was very sensitive to substitution by both 19:0 and 18:3. Although not conclusive, these results suggest that alterations of the fluid state of the membrane that affect lateral movements of surface proteins may not affect cytolytic processes.
The Requirement for the Fc Portion of Antibody in Antigen-Antibody Complex-Mediated Suppression
Authors: Morgan Edward L. , Tempelis Constantine H.
Published: January 2023

The role of the Fc portion of antibody in immune complex-induced suppression was studied in vivo and in vitro. BSA pheasant anti-BSA complexes, formed in antigen excess (Ag1Ab1), were found to suppress both responses to BSA and SRBC. When complexes were formed with F(ab′)2 fragments of pheasant anti-BSA, no suppression was observed, indicating that the Fc piece was indeed essential for the induction of Ag-Ab complexmediated suppression.
Studies on the Terminal Stages of Immune Hemolysis
Authors: Boyle Michael D. P. , Langone John J. , Borsos Tibor
Published: January 2023

The intermediate product EAC1-8 released cytoplasmic components as a result of at least two sequential reactions after its interaction with C9. Binding of C9 to EAC1-8 occurred in a few minutes even at 0°C. Trypsinization of EAC1-9 prepared and held at low temperature resulted in nullification of the potential hemolysis of these cells. A brief incubation at 30 or 37°C resulted in the formation of an intermediate whose hemolytic potential could not be nullified by trypsin. The failure of trypsin to nullify hemolysis was attributed to the insertion of C9 into the cell membrane. Studies on the effect of EDTA or low temperature suggested that the reported temperature-dependent step in E* formation described by Frank et al. was the insertion of C9. The results of the studies with 86Rb-labeled EAC1-8 indicated that a transmembrane channel was not formed until after the C9 had been inserted and a further reaction or reactions had occurred.
Genetic Control of the T Lymphocyte Proliferative Response to Staphylococcal Nuclease: Evidence for Multiple MHC-Linked Ir Gene Control
Authors: Schwartz Ronald H. , Berzofsky Jay A. , Horton Clare L. , Schechter Alan N. , Sachs David H.
Published: January 2023

Genetic control of the T lymphocyte proliferative response to staphylococcal nuclease was investigated with the sensitive peritoneal exudate, T lymphocyte enriched cells (PETLES) assay. All mouse strains examined responded to nuclease when immunized with large doses (0.6 nmole) of the antigen in complete Freund's adjuvant. Nevertheless, control of responsiveness by major histocompatibility (MHC)-linked immune response (Ir) genes was discerned when the nuclease responses were normalized to the PPD response of the same strain and compared among strains possessing the same non-H-2 background. In addition, lowering the immunization dose enhanced the difference between low and high responder strains. Genetic mapping studies carried out under these limiting conditions confirmed the earlier findings of an Ir-Nase gene mapping to the I-B subregion of the H-2 complex on the basis of low responsiveness by the B10.A(4R) and B10.A(5R) recombinant strains. However, immunization of these recombinant strains with 0.6 nmole of nuclease demonstrated a difference in their responsiveness, suggesting that the genetic control might be more complex. This suggestion was confirmed by cross-stimulation studies of nuclease-primed PETLES with fragments of nuclease. The pattern of the proliferative responses was found to be controlled by MHC-linked genes mapping in the I (and possibly the K and D) region, and appeared to be unique for each haplotype. In the case of the 4R and 5R recombinants the patterns appeared different from either that for the high responder B10.A or that for the low responder B10 parental strains. These results suggest that several Ir genes are involved in the T cell proliferative response to nuclease. Furthermore, the fact that fragments of nuclease, which possess little of the tertiary structure of the native molecule, could stimulate a proliferative response in PETLES immunized to whole nuclease suggests that T cells, in contrast to most antibodies, recognize antigenic determinants that possess a similar conformation in both the native molecule and the fragments.
Absence of Correlation of Ig-1 Allotype and IgE Antibody Suppression in SJL Mice
Authors: Itaya Takaki , Okumura Ko , Watanabe Naohiro , Ovary Zoltan
Published: January 2023

In previous studies it has been found that in the SJL strain of mice, normal thymus-derived cells have a suppressor effect on anti-hapten IgE production. It has also been demonstrated with (SJL × BALB/c)F1 mice and backcrosses to the SJL parent that the suppressive function is inherited by a single autosomal Menedelian gene, not linked to the H-2 gene complex (1).

Chronic allotype suppression can be obtained in (SJL × BALB/c)F1 progenies if the BALB/c female before mating is immunized with Ig-lb (paternal, i.e., SJL) allotype (2, 3). In the suppressed (SJL × BALB/c)F1 mice IgG2a immunoglobulin subclass can be detected, but only of the Ig-la allotype and not of the Ig-lb allotype. It is well known that the IgG2a immunoglobulin subclass carries the Ig-la and Ig-lb allotypes.

It was of interest, therefore, to examine if in the sera of the backcross mice (SJL × BALB/c)F1 × SJL a correlation could be found between the nonpersistence of anti-hapten IgE production and the Ig-Ib allotype.
Association of C8 Deficiency with Sickle-Thalassemia Hemoglobinopathy
Authors: Agnello Vincent , Pariser Kenneth M. , Babott Doreen
Published: January 2023

Three pedigrees with hereditary C8 deficiency have thus far been reported. All three propositi have been black. Two additional cases of selective C8 deficiency are reported here, a black female with complete deficiency of C8 and a black male with approximately 50% of the C8 normal mean. Both have sickle-thalassemia hemoglobinopathy (S-T) with virtually identical percentages of A2, F and S hemoglobin. The female has two offspring that carry only the sickle trait and have normal C8 levels. Family studies have not been possible in the second case. Four additional patients with sickle-thalassemia hemoglobinopathy have been thus far studied. All have normal C8 levels, but their hemoglobin patterns while diagnostic for S-T disease differ significantly from the first two cases studied. The C8 levels in several cases of sickle cell, sickle trait, thalassemia, and thalassemia trait disease, thus far studied have been normal.
Lack of C5 Convertase-Generating Activity in Naja haje Cobra Factor
Authors: Bauman N.
Published: January 2023

Commercial cobra factor is prepared from the venom of either Naja naja, the Indian cobra, or Naja haje, the Egyptian cobra, and is not marked as to source. Cobra factor from Naja haje (CVFh), although able to generate C3-cleaving activity, does not support passive lysis. Cobra venom from Naja naja (CVFn), upon incubation with normal human serum, depletes C3 through C9, and causes conversion of C3 and B as observed in immunoelectrophoresis. CVFh also causes conversion of C3 and B and depletes C3; but C5 through C9 are spared. CVFh and CVFn were found to be equally resistant to C3bINAhu.

When CVFn was incubated with normal human or guinea pig serum, an activity was generated which was detectable as a zone of hemolysis in agarose plates containing guinea pig erythrocytes and EDTA, when placed near a well containing human or rat, but not guinea pig, CEDTA.
Regulation of Macrophage Motility by Components of the Complement System
Authors: Bianco C. , Götze O. , Cohn Z. A.
Published: January 2023

Inhibition of macrophage migration is a well established assay for delayed hypersensitivity. The inhibitory factor (MIF) is a lymphokine which also plays a role in macrophage activation. In this report we show that complement derived peptides can regulate the motility of macrophages in a MIF type assay. Cleavage products of factor B inhibit migration, while cleavage products of C5 increase macrophage migration.

Mouse peritoneal cells obtained 5 days after i.p. injection of mineral oil were washed and centrifuged into capillary tubes. The capillaries were sectioned at the medium-cell interface and placed into chambers containing 450 µl of 10% heat-inactivated fetal bovine serum (56°C, 1 hr) in RPMI 1640 medium. After addition of the complement derived peptides, the chambers were incubated for 18 hr at 37°C and the areas of migration determined. More than 90% of the cells remained viable at the end of the assay.
Affinity Labeling of C1s̄ with an Extended Reagent
Authors: Bing David H. , Laura Richard , Cory Michael
Published: January 2023

The reagent m-[o-(2-chloro-5-sulfonylfluorphenylureido) phenoxybutoxybenzamidine [mCP(PBA)-F] is an extended affinity labeling reagent in which the reactive sulfonylfluoride is 17.1 Å from a cationic benzamidine function that competitively inhibits C1s̄ esterase and protease activity. Purified human C1s̄ with high specific esterolytic activity was selectively and irreversibly inactivated with mCP(PBA)-F. At 16.6 µM mCP(PBA)-F and 22.1 µM C1s̄, the time course of inactivation followed pseudo first order kinetics (kL = 2.45 × 10-3 sec-1, 23°C). The initial rate of inactivation of C1s̄ at several mCP(PBA)-F concentrations followed a rectangular hyperbolic function preducted for affinity labeling kinetics (KL = 87.3 µM, k2 = 3.02 × 10-3 sec-1). The hydrolyzed affinity labeling reagent lacking the reactive sulfonylfluoride group [mCP(PBA)-OH] was a strong competitive inhibitor (KI = 6.82 µM vs 0.63 mM for benzamidine alone) implicating active site binding.
Is Factor D of the Alternative Pathway a Fragment of Thrombin?
Authors: Davis A. E. , Rosenberg R. D. , Fenton J. W. , Bing D. H. , Rosen F. S. , Alper C. A.
Published: January 2023

Properdin factor D (GBGase, C3Pase) is a 25,000 dalton protease required for alternative pathway activation. After isoelectric focussing of normal human serum (NHS) in polyacrylamide gel, an agar gel overlay containing 1.7% guinea pig erythrocytes and 10% NHS in 0.05 M barbital buffer with 1 mM mg++ and 5 mM EGTA developed lysis bands for C3, factor B and one other protein with a more basic isoelectric point. The latter (but not bands of C3 or B) is also produced in an overlay containing factor D-depleted serum instead of NHS (Martin et al.). In both these assays, purified human α, β/γ, and autocatalyzed thrombin (T) produced lysis bands corresponding to protein distribution. The major band of autocatalyzed, but no other T, coincided with factor D. Incubation of β/γT in NHS resulted in no noticeable loss of intensity of the lytic bands.
Activation of the Alternative Complement Pathway by Escherichia Coli: Resistance of Bound C3b to Inactivation by C3bINA and β1H
Authors: Fearon D. T.
Published: January 2023

The surfaces of activators of the alternative pathway, such as zymosan and rabbit erythrocytes, provide sites which protect bound C3b from inactivation by C3b inactivator (C3bINA) and β1H, and P,C3b,Bb from decay-dissociation by β1H. This circumstance permits surface-associated amplified generation and deposition of C3b. The capacity of a bacterial surface, if analogous to zymosan and rabbit erythrocytes, to protect and thereby accumulate increasing amounts of C3b by local circumvention of the regulatory proteins would be compatible with a unique role of the alternative pathway in host defense. The susceptibility of C3b bound to Escherichia coli to C3bINA was compared with that of C3b bound to the nonactivator, sheep erythrocytes, in the state, EAC4b,3b. E. coli W3110, killed by heating at 60°C for 60 min, activated the alternative pathway in human serum diluted 1:5 in GVB containing 2 mM Mg++ and 8 mM EGTA as demonstrated by 82% C3 and 87% B consumption.
Purification and Characterization of a Mouse Serum Protein with Specific Binding Affinity for C4 (Ss protein)
Authors: Ferreira Arturo , Weisz-Carrington Paul , Gigli Irma , Nussenzweig Victor
Published: January 2023

A new protein named Ss-bp, or C4-bp, with specific binding affinity for activated C4 has been found in mouse serum. Ss-bp is a heat-stable (60° C, 1 hr) β-globulin with a sedimentation coefficient of 10S. Ss and Ss-bp are separated by filtration of EDTA-plasma in Sephadex G-200. However, in serum, Ss-bp binds tightly to Ss and the complex is found in the excluded volume of the column. After electrophoresis in agarose at pH 8.6, the Ss-bp/Ss complex moves faster toward the anode than either protein alone. For this reason Ss-bp may appear to be polymorphic in serum of mice with high levels of C4 (Ss-H) versus those with low levels of C4 (Ss-L) (Ferreira, Takahashi and Nussenzweig, J. Exp. Med., in press).

Ss-bp levels are higher in serum of males (160 µg/ml) than in female mice (60 µg/ml). As in the case of other mouse C components (C4, C5), the concentration of Ss-bp in serum is markedly influenced by testosterone.
The Structural Events Associated with the Attachment of Complement Components to Cell Membranes in Reactive Lysis
Authors: Dourmashkin R. R.
Published: January 2023

The events occurring at the cell membrane during reactive lysis were studied by electron microscopy, using purified complement components (generously donated by Drs. W. Kolb, E. Podack and H. J. Müller-Eberhard, Scripps Clinic). At the stage, no particles were seen attached to the membrane after preparation for E.M. At the stage, a foliaceous particle was seen attached to the membrane surface, with a narrow stalk and two to three arms extending outward. At the stage, the particles appeared larger and possessed up to six arms. None of these stages showed any lesions previously described as “complement holes.” However, the stage showed many typical complement lesions, presumably due to the rearrangement of the structure of the particles.

The lesions in profile appeared to show channels that penetrated all or part of the thickness of the cell membrane; however, such observations must be interpreted with caution, as the relation of the E.M. negative staining image of the membrane to the biologic structure has not been clarified.
Properties of the Early Classical Complement System of Mice
Authors: Goldman John N. , Goldman Margaret B.
Published: January 2023

Mouse serum and EDTA plasma were subjected to low ionicity precipitation, ion exchange chromatography and gel filtration in an attempt to separate and purify C1, C4 and C2. Although they could be obtained free of many other serum proteins these three components could not be significantly separated from one another using a wide variety of ion exchange resins and gels and conditions of elution. C1, C4 and C2 eluted from Sephadex G-200 in buffers containing EDTA with an apparent molecular weight of approximately 150,000 and in buffers containing Ca++ and Mg++ with an apparent molecular weight of over 500,000. Cell bound activity can be formed by incubation of EA in mouse serum as indicated by the lysis of these cells upon the later addition of guinea pig serum in EDTA. Incubation of EA in mouse serum followed by incubation in buffers containing 10 mM EDTA resulted in a rapid loss of all detectable C1, C4 and C2 functional activities.
The Induction of Macrophage Spreading by Factor B of the Properdin System
Authors: Götze O. , Bianco C. , Cohn Z. A.
Published: January 2023

The previously reported complement-dependent spreading of freshly explanted mouse peritoneal macrophages was further analyzed. Whereas the screening of components of the classical pathway did not reveal any activity, mixtures of human Factors B, D̄ and C3b (after incubation) induced spreading in 50–80% of 5 × 104 glass-attached macrophages within 60–120 min. In order to define the active factor(s) or their fragments 125I-labeled B was added to fresh human serum which was then activated for 60 min, 37° using 5 mg inulin/ml serum. After fractionation of the serum on DEAE- and CM-cellulose and on Sephadex G150 columns spreading activity was found in fractions containing the activated form of B (Bb) and closely followed the distribution of 125I-Bb in the columns' eluates. Fractions containing C3b or Ba lacked activity. Highly purified Bb was prepared from purified B by incubation with D̄, and C3b. Bb was then separated from D̄, Ba and C3b by DEAE-cellulose chromatography.
Differential Patterns of C3b Receptor Expression on Guinea Pig Macrophages
Authors: Koopman W. J. , Wahl L. M. , Sandberg A. L. , Mergenhagen S. E.
Published: January 2023

Initial investigations revealed that the expression of C3b receptors on macrophages was markedly influenced by the manner in which the cells were obtained. Resident (wash-out), glycogen-induced and oil-induced guinea pig peritoneal cells were harvested and adherent cell monolayers were cultured in a serum-free Dulbecco's medium at equivalent cell concentrations. Monolayers contained 90 to 95% macrophages as assessed by nonspecific esterase activity and latex ingestion. Macrophage membrane C3b receptors were detected by rosetting with EIgMC143bgp indicator cells (100 IgM sites/indicator cell; 20 RBC/macrophage) at varying times after initiation of culture. Marked differences in C3b receptor expression were observed among the three macrophage populations. 81%±4% of resident peritoneal macrophages rosetted with EIGMC143bgp indicator cells at the initiation of culture and minimal decline in C3b receptor expression occurred at 16 hr of culture (70%±5% rosettes). In contrast, 35%±8% of oil-induced peritoneal macrophages rosetted initially and by 16 hr only 15%±9% of these cells formed rosettes with C3b-bearing indicator cells.
Monocyte Complement Stimulator (M.C.S.): A T Lymphocyte Product Capable of Stimulating Production of the Second Complement Component (C2)
Authors: Littman Bruce H. , Ruddy Shaun
Published: January 2023

We have previously reported that the production of the second complement component (C2) by human monocytes can be stimulated by co-culture with lymphocytes and antigen or with lymphokine-rich culture supernates. Using lymphocyte subpopulations separated on the basis of rosetting with sheep erythrocytes (T-lymphocytes), we now report that only cultures composed of antigen-stimulated monocytes and T lymphocytes could produce this lymphokine activity. Macrophage migration inhibitory factor (M.I.F.) was also present in this supernate. Although antigen-stimulated B lymphocytes (rosette-negative lymphocytes), B lymphocytes with monocytes, monocytes only, and T lymphocytes only could not produce this lymphokine activity, supernate of antigen-stimulated B lymphocytes and monocytes did contain M.I.F., indicating that molecules with this activity are not necessarily identical with those responsible for stimulating C2 synthesis by monocytes.

Further, Sephadex G-100 gel filtration of supernates from antigen-stimulated lymphocyte cultures resulted in fractions also capable of stimulating monocytes to increase their C2 production.
Normal and Chronic Lymphocytic Leukemia Lymphocytes As Targets for Human Complement
Authors: Logue G. L. , Huang A. T.
Published: January 2023

Human lymphocytes are resistant to cytolytic effects of human complement in vitro. In order to study this phenomenon, the amount of human third component (C3) bound to lymphocytes from normal subjects and patients with chronic lymphocytic leukemia (CLL) was determined when complement was activated by reacting lymphocytes with anti-I antibody, anti-HLA-2 antisera, and rabbit anti-human lymphocyte antisera. C3 was quantitated by an anti-C3 consumption assay. Human C3 was bound to both normal and CLL lymphocytes in a dose-response fashion when the cells were exposed to human serum and the anti-lymphocyte antibodies. No quantitative differences were observed in binding of C3 to normal and CLL lymphocytes. Cytolytic effects of complement activation were measured by cell counting, dye exclusion, 51Cr release, release of 3H-thymidine labeled DNA, and the ability of the cells to incorporate 3H-thymidine and 3H-uridine into DNA and RNA.
A Role for IgG in the Activation of the Human Alternative Pathway by Rabbit Erythrocytes
Authors: Nelson Ben , Ruddy Shaun
Published: January 2023

A serum factor which participates in the early steps of the lysis of rabbit erythrocytes (E) by human serum in Mg-EDTA has been partially characterized. Normal human serum (NHS) when adsorbed with rabbit E or stromata at 0°C in the absence of divalent cations, lost 35% of its hemolytic activity against fresh rabbit E. The feedback loop activity (B, D, P) of the adsorbed serum was intact. The lost hemolytic activity was restored with a pH 3.4 eluate from the adsorbed stromata; the activity in the eluate has been termed “0° factor E.”

When 0° factor E was added to serum depleted of 0° factor E by prior absorption with rabbit E, a linear dose response was obtained which was concave to the abscissa, indicating that a single activity was being added. The activity was unaffected by heating to 56°C for 30 min and had an apparent molecular weight of 700,000 on Sepharose 4B gel filtration.
Modulation of the Alternative Pathway in Dilute Whole Serum by Introduction of Purified Components and Control Proteins
Authors: Nydegger U. , Fearon D. T. , Austen K. F.
Published: January 2023

Activators of the alternative pathway of complement such as zymosan, rabbit erythrocytes, and heat-killed E. coli protect bound C3b from C3bINA and P, C3b, Bb from decay-dissociation by β1H, thereby amplifying C3b generation and deposition. The capacity of purified components and control proteins to modulate alternative pathway activation by zymosan and rabbit erythrocytes (Er) was assessed in dilute C2-deficient human serum.

Zymosan-induced inactivation of C3 and B was determined at different intervals after 100 µg of zymosan were added to 150 µl of serum diluted 1:6 in VBS++. The mean inactivation of C3 and B at 45 minutes was 61 ± 5.4% (±1 SD) and 62 ± 5.7%, respectively. Various amounts of the control proteins were added to the serum to determine the increase over endogenous concentrations necessary to inhibit 50% of the zymosan effect. Increases of C3bINA by 43% and of β1H by 25% inhibited 50% of zymosan-induced inactivation of C3.
Antibody Responses to a T Cell Dependent Antigen in C4 Deficiency
Authors: Ochs Hans D. , Jackson Charles G. , Frank Michael M. , Hosea Stephen W. , Wedgwood Ralph J.
Published: January 2023

Complete absence of the fourth component of complement has been described in man and in guinea pigs. Recently, a close linkage between the gene(s) controlling synthesis of C4 and the major histocompatibility complex has been demonstrated in man as well as in the guinea pig.

Because of a possible relationship between immune responses and the complement system, we have studied antibody responses to a T cell dependent antigen in a patient and in guinea pigs with C4 deficiency. Bacteriophage Φχ 174, given intravenously, was cleared within one week after primary injection, similar to normal controls. The primary antibody response was markedly suppressed and shortened: in normal controls, antibody titers (mean Kv > 8) persisted for over 4 weeks; in C4 deficiency, antibody (peak titer < 1) disappeared 2–3 weeks following immunization. In the normal control population, a second injection of antigen resulted in a rapid rise of antibody (Kv > 100) and transition from IgM to IgG.
Complement Cleavage Products in Inflammatory Exudates from Patients with Periodontal Diseases
Authors: Schenkein Harvey A. , Genco Robert J.
Published: January 2023

Periodontal diseases are characterized by an inflammatory exudate termed gingival fluid (GF). We have examined selected complement components for cleavage products in GF from patients with periodontitis and the less common disease, periodontosis. We hope to establish an objective criterion for diagnosis as well as to provide information on the mechanisms involved in producing periodontal inflammation.

GF samples (2 µl) were collected from 18 patients diagnosed as having severe periodontitis and blood samples were collected simultaneously. Serum and GF were stored at -76°C until assayed. Protein concentrations were measured by single radial immunodiffusion for nine plasma proteins, including C3, C4, and Factor B. An antiserum to C3 specific for the B determinant was utilized. It was found that GF contained only 20% of the a-C3 (B determinant) reactive material found in serum, while all other proteins measured were at levels 70 to 80% of that in serum.
Stimulation of Synthesis and Release of Lipids in Tumor Cells Under Attack by Antibody Plus Complement
Authors: Schlager Seymour I. , Ohanian Sarkis H. , Borsos Tibor
Published: January 2023

Resistance to antibody-complement- (Ab-C) mediated killing of guinea pig hepatoma cells (line-1 and line-10) correlates with the ability of the cells to synthesize lipids, but not DNA, RNA, protein, or complex carbohydrate. We therefore studied the synthesis and release of macromolecules (especially lipids) by tumor cells under attack by Ab plus C.

Ab-sensitized tumor cells (TA) incorporated increased amounts of acetate, glycerol, or fatty acids into complex lipids compared to control cells within 5 to 10 min after the addition of guinea pig C (GPC) to the cells (TAC). TA, TC, or TAΔC (Ab plus heat-inactivated GPC) did not show this effect. No enhancement of DNA, RNA, or protein synthesis in TAC as compared with control cells was noted. Line-1 cells, which are killed by GPC when sensitized with excess anti-Forssman Ab (TAxs), demonstrated enhanced lipid synthesis within 1 to 3 min after the addition of GPC to TAxs, before measurable killing of the cells had occurred.
Interaction of the First Component of Complement in Platelet Adhesion and Aggregation Induced by Collagen and Aggregated IgG
Authors: Wautier J. L. , Souchon H. , Reid K. B. M. , Peltier A. P. , Caen J. P.
Published: January 2023

Pure C1 subcomponents (C1q, C1r, C1s) and fragments were obtained by limited proteolysis with collagenase and pepsin. The fragments prepared with pepsin contained the collagen-like region of C1q whereas the collagenase-treated C1q had lost its collagen portion.

The different C1 subcomponents or fragment of C1q were tested for their effect on human platelets and on collagen-induced aggregation and adhesion of gel-filtered platelets. The collagenase-treated C1q was without effect in platelet collagen interaction.

The pepsin-derived fragment of C1q containing the collagen-like region inhibited platelet aggregation and adhesion to collagen. The preincubation of C1s with collagen inhibited platelet adhesion and aggregation. Studies on platelet aggregation induced by aggregated IgG showed that the complete molecule of C1q is necessary for the reaction.

The results suggest that the collagen-like region of C1q reacts with the platelet, and possibly with platelet-associated C1s or a structure similar to C1s.
Stability of DNA/Anti-DNA Complexes
Authors: Taylor Ronald P. , Weber Daniel , Broccoli Anthony V. , Winfield John B.
Published: January 2023

Antigenic specificity, concentration in serum, and binding avidity of different types of antibodies to DNA all contribute to the nature of circulating DNA/anti-DNA complexes in systemic lupus erythematosus (SLE), and thereby influence the potential of such complexes for tissue injury in this disorder. With highly purified single(s) and double(d) stranded 3H-DNA prepared from Escherichia coli, the rate of dissociation of DNA/anti-DNA complexes in the presence of excess unlabeled DNA was studied as a function of temperature and DNA conformation. At cold temperatures dissociation of complexes of antibody and ssDNA or dsDNA was slow and did not reach equilibrium. At 37°C “off rates” for ssDNA/antibody complexes generally were rapid, dissociation being complete within 1 hr. dsDNA/antibody complexes, on the other hand, were much more stable at higher temperatures than those formed with ssDNA. With many SLE sera, the majority of dsDNA remained bound to antibody after 24 hr of incubation with a large excess of unlabeled DNA. Kinetic studies of this type were compared with binding isotherms as methods of obtaining information concerning binding avidities of antibody for dsDNA and ssDNA.

The implications of these findings are discussed with regard to i) the pathogenicity of dsDNA/anti-DNA complexes, and ii) the difficulty of estimating binding avidity in multivalent antigen, antibody systems.
Damage to Schistosomula of Schistosoma Mansoni Induced Directly by Eosinophil Major Basic Protein
Authors: Butterworth Anthony E. , Wassom Donald L. , Gleich Gerald J. , Loegering David A. , David John R.
Published: January 2023

Major basic protein (MBP) from both human and guinea pig eosinophils has been found to damage schistosomula of Schistosoma mansoni at concentrations as low as 2 × 10-5 M. This damage can be detected both by light microscopy and by measurement of release of 51Cr from labeled larvae. Damage is attributable in part to the basic properties of the molecule, since some other cations of comparable molecular weight, such as protamine and polyarginine, have comparable effects at equimolar concentrations. Other cations, however, fail to mediate damage, indicating that simply the possession of a positive charge is a necessary but insufficient prerequisite. The action of MBP is not selective, since mammalian target cells can also be damaged. Damage induced by MBP is associated with uptake of the material onto the parasite's surface, and both uptake and damage can be blocked by heparin.

In addition, it has been found that MBP is released during the antibody-dependent reaction between human eosinophils and schistosomula, and can be detected both on the surface of the organism and free in small quantities in the supernatant. Damage mediated directly by eosinophils is not inhibitable by heparin, suggesting that the effect of eosinophils is attributable to the local release of high concentrations of MBP into a sequestered site adjacent to the parasite surface, rather than to a generalized release into the supernatant.

These findings suggest that release of MBP may represent a major mechanism of eosinophil-mediated damage to schistosomula and, since MBP is uniquely associated in high quantities with the eosinophil, these results provide a partial explanation for the selective action of eosinophils on antibody-coated schistosomula.
Functional Studies on Lymphocytes in Adult Human Bone Marrow
Authors: de Gast Gÿsbert C. , Platts-Mills Thomas A. E.
Published: January 2023

Bone marrow (BM) was obtained from rib sections removed during thoracotomy on 10 patients (mean age 45 years) with a primary lung tumor. A linear sucrose-serum density gradient was used to fractionate the nucleated bone marrow cells. The pellet fraction, which contained only a few lymphocytes but nearly all the morphologic plasma cells, showed spontaneous IgG and IgM production. The upper fraction (5 to 8% sucrose), comprising 82% lymphoid cells (24.2% T cells and 14.0% B cells (SmIg+ve cells)) showed no spontaneous Ig production, but in five cases produced Ig when stimulated with Pokeweed mitogen (PWM). After T/non-T cell separation of this lymphocyte rich (upper) fraction, the BM-non-T cells produced little or no Ig on their own even when they were stimulated with PWM. However, these cells produced IgM (mean 0.57 μg/ml) and/or IgG (mean 1.69 μg/ml) when cultured with mitomycin-C-treated allogeneic peripheral blood T cells (PB-Tm) and PWM. Parallel cultures of similarly separated PB-non T cells in the presence of PWM and the same PB-Tm produced significantly more Ig (mean IgM 6.02 μg/ml; mean IgG 5.33 μg/ml). When BM-T cells were cultured with PB-non-T cells they appeared to give very poor help for Ig production (IgM mean 0.07 μg/ml, IgG mean 0.27 μg/ml). The results show that bone marrow lymphocytes as judged by Ig production are functionally poor compared with peripheral blood lymphocytes. It seems possible that at least part of the BM-B cells are immature and not able to respond to PWM. The T cells in bone marrow may also be immature or may belong to T cell subpopulations other than helper T cells.
Demonstration of A Rabbit Cell Surface Ig That Bears Light Chain and VH, But Lacks µ-, α-, and γ-Allotypes-Rabbit IgD?
Authors: Wilder Ronald L. , Chew Yuen Catherine , Coyle Sharon A. , Mage Rose G.
Published: January 2023

Based on previous biochemical studies of rabbit sIg, we postulated that if rabbits possessed an IgD-like molecule, its heavy chain might co-electrophorese with the µ-heavy chain on reduced SDS-PAGE. Accordingly, we studied Ig from rabbit spleen cells radiolabeled biosynthetically or by cell surface iodination. Cell lysates were prepared and immunoprecipitation studies were performed by standard techniques. Sequential absorptions in which total removal of µ-allotype (n-locus) was followed by immunoprecipitation with anti-a(VH) or anti-b (kappa) showed residual band(s) with µ-relative mobility on SDS-PAGE under reducing conditions. Similar immunoprecipitations with anti-gamma (γ) or anti-alpha (α) reagents failed to precipitate the residual band with µ-mobility. The residual band was found in both radioiodinated and biosynthetically radiolabeled lysates.

When direct immunoprecipitation was carried out in the absence of proteolytic enzyme inhibitors, the SDS-PAGE pattern of total Ig under reducing conditions frequently showed three peaks, corresponding to µ (MW ∼70,000), light chain (MW ∼23,000), and an unidentified heavy chain (MW ∼65,000). The second heavy chain peak was not immunoprecipitated with µ, γ, or α allotype sera. Thus, we have identified a “new Ig” in the rabbit that bears VH and light chain, and lacks µ, γ and α allotype markers. The molecule appears to be proteolytically labile, but has an undegraded heavy chain that co-electrophoreses with µ-chains on reduced SDS-PAGE. The similarities to published data on human and rat lymphocyte IgD strongly suggest, but do not formally prove, that we have identified the rabbit homologue of IgD.
Bullous Pemphigoid Antigen
Authors: Diaz Luis A. , Patel Harish , Calvanico Nickolas J.
Published: January 2023

This report concerns the isolation of bullous pemphigoid antigen from the nondialyzable urinary components of a patient with the disease. The isolation was accomplished by ion exchange chromatography and gel filtration. Pemphigoid antigen was found to be a basic glycoprotein that on SDS gel electrophoresis showed two major bands, one in the 18,000 m. w. region and the second with a m. w. of 74,000. Between these two bands, two additional bands appeared; one of 35,000 daltons and the other of 68,000 daltons. The 18,000 m. w. band was eluted from the gel and rerun on SDS gels. These gels showed the 18,000 m. w. band and also the appearance of the 35,000 and 74,000 m. w. bands. This finding indicates that urinary pemphigoid antigen may exist both as a single monomeric form and in polymeric aggregates.
Virus Specificity of Human Influenza Virus-Immune Cytotoxic T Cells
Authors: Biddison William E. , Shaw Stephen , Nelson David L.
Published: January 2023

The virus specificity of human in vitro cytotoxic T cell responses to influenza virus was studied with the use of peripheral blood mononuclear leukocytes from normal adult volunteers. Previous natural exposure of these donors to a variety of type A influenza viruses was documented by HI antibody titers. Cells sensitized in vitro with A/HK or A/PR8 were cytotoxic for autologous target cells infected with A/HK, A/PR8, or A/JAP 305 type A influenza viruses, but not for B/HK-infected or uninfected cells. B/HK-sensitized effector cells lysed target cells infected with B/HK but not targets infected with type A viruses. A/HK- and A/PR8-immune effector populations were shown to recognize cross-reactive antigens on A/HK- and A/PR8-infected target cells by cold target competition. Influenza-immune effector cells were cytotoxic for virus-infected autologous targets but much less so for virus-infected allogeneic targets. This self-restriction suggested that the cytotoxicity was largely T cell-mediated and was confirmed by cell separation analysis.

Thus, the human secondary cytotoxic T cell response in vitro to influenza viruses is predominantly directed against cross-reactive determinants on cells infected with serologically distinct type A influenza viruses.
Antigenic Specificities of the Cellular Immune Response of C57BL/6 Mice to the Moloney Leukemia/Sarcoma Virus Complex
Authors: Enjuanes Luis , Lee John C. , Ihle James N.
Published: January 2023

The antigenic specificities of the cellular immune response against MoLV-MSV-induced tumors in C57BL/6 mice were studied by using a variety of purified MoLV virion proteins. Cellular reactivity, as assessed by the ability of purified proteins to induce T cell proliferation, was primarily directed against the virion envelope glycoprotein and to a lesser extent against p12. No blastogenesis was detectable with the viral p10, p15, or p30 proteins. The optimal gp71 and p12 antigen concentrations required for 50% of maximal responses were 0.036 and 0.013 nmoles per 5 × 105 cells per 0.2 ml, respectively. Blastogenesis was dependent upon the presence of T cells, but was apparently independent of the presence of macrophages.

The antigenic specificity of cellular cytotoxicity was assessed by microcytotoxicity assays by using transformed nonproducer and producer cell lines with a various virus serotypes. The results demonstrated that cytotoxicity was immunologically type-specific for MoLV serotype of virus and is dependent upon the expression of the MoLV viral envelope glycoprotein gp71. The requirement for gp71 cytotoxicity was further assessed by antigen-blocking experiments. These results demonstrate that only MoLV gp71 could significantly block cytotoxicity (τ80%), whereas no blocking was seen with RLV gp71, MoLV p30, p15, p12, or p10, suggesting that the major cytotoxic response is directed against MoLV gp71. The temporal relationships of the cellular as well as the humoral immune responses related to tumor development and regression are also described.
Increased photosensitivity to near-ultraviolet light in murine SLE.
Authors: Golan D T , Borel Y
Published: January 2023

We investigated whether there is increased susceptibility to near-UVL in murine SLE. Cultured spleen cells from either strain of mice with lupus disease or conventional strains of mice were exposed to different UVL fractions in vitro. The effect of DNA synthesis, release, and repair was examined. DNA synthesis and release was measured as percent of [3H]thymidine (dT) uptake into either total acid-precipitable radioactive material of cell sediment plus supernatant, or that of the medium alone, whereas hydroxyurea-resistant dT incorporation represented DNA repair. The data indicate that all SLE strains, in contrast to all non-SLE strains, show increased DNA synthesis and release after UV-A exposure. In addition, all murine SLE strains demonstrate increased susceptibility to induction of DNA damage by UV-A. The significance of these observations in relation to the clinical activity of SLE after sunlight exposure is discussed.
Regulation of TCGF production in T cells. II. Early membrane events after anti-Thy-1 binding by the TCGF-producing T lymphoma EL-4 G-12.
Authors: Norcross M A , Smith R T , Shimizu S
Published: January 2023

The T cell lymphoma line EL-4 G-12 produces large quantities of TCGF (IL 2) shortly after exposure to anti-Thy-1 and cross-linking reagents. Early surface events that initiate nonconstitutive synthesis and secretion have been examined by fluorescent microscopy, EM analysis, flow cytometry of anti-Thy-1, and measurements of membrane polarization. The data show that anti-Thy-1 binding leads to immediate and rapid endocytosis and shedding of the antibody molecules, but with no change in membrane potential. Cross-linking of the anti-Thy-1 results in stabilization and accumulation of the antibody on the surface, and rapid hyperpolarization. The data suggest that cross-linking of ligand antibody interactions at the surface is a regulatory event involved in initiation of the signal for TCGF synthesis and secretion.