Spleen Fragments Research Articles

Homogeneous antibodies directed against human cell surface antigens: I. The mouse spleen fragment culture response to T and B cell lines derived from the same individual

The use of the mouse spleen fragment culture system is extended to the production of antibodies to human lymphoblastoid cell lines. These antibodies were tested for reactivity against the immunizing cell line, and against a second cell line which had been derived from the same human blood sample. Many of the antibodies were found to discriminate between the 2 isogenic lines. These results demonstrate the potential of the mouse spleen fragment culture system to provide homogeneous reagents which detect distinguishing markers on closely related human cells.

IMMUNOLOGICAL MEMORY AFTER PRIMING WITH A THYMUS INDEPENDENT ANTIGEN, NIP‐FICOLL

The capacity of mouse spleen fragments to mount an anti-NIP (4-hydroxy-5-iodo-3-nitrophenylacetyl) response in vitro was studied. The fragments came from unprimed, NIP-Ficoll (polymer of sucrose and epichlorhydrin) or NIP-CG (chicken globulin) primed mice. Unprimed spleen fragments from C57BL/6 mice gave a good anti-NIP response to NIP-Ficoll, whereas CBA fragments did not. Priming with NIP-Ficoll made CBA fragments responsive and enhanced slightly the response of C57BL/6 fragments when stimulated with the same antigen. This memory effect could be seen only after a small priming dose. Priming the mice with NIP-Ficoll made their spleen fragments responsive to a protein conjugate of NIP (NIP-CG), but this effect was seen only after priming with a high dose. The antibody class distribution and the kinetics of the appearance of different immunoglobulin classes were similar in the primary and secondary responses in vitro. The peak responses of IgM, IgA and IgG were reached on day eight and the relative amount of IgG was the same in the primary and in the secondary responses. Spleen fragments derived from NIP-CG primed mice produced more IgG anti-NIP antibodies than fragments derived from untreated mice when immunized in vitro with NIP-Ficoll. The amount of IgG was, however, much higher when these fragments were challenged with the homologous antigen, NIP-CG.

The solubilization of HLA antigens with detergents and partial characterization of the antigen-detergent complexes

The A and B antigens specified by loci within the human HLA genetic complex have been solubilized with sodium deoxycholate and Triton X-100 from the membranes of lymphoblastoid cell lines and surgically-obtained human spleen fragments. These detergents have been evaluated in terms of their cytolytic effects on viable cell suspensions and their ability to solubilize both total membrane protein and HLA serologic activity. Both detergents were effective reagents for the solubilization of HLA serologic activity when used above their critical micelle concentrations (CMC). Aggregation of deoxycholate solubilized HLA antigens occurred when the concentration of the detergent was adjusted to concentrations below the CMC. Hydrodynamic parameters for HLA-A and B-deoxycholate complexes have been evaluated by column chromatography and sucrose density-gradient centrifugation. The predominant molecular species for both HLA-A and B-deoxycholate complexes were characterized as having Stokes' radii of 41 ± 1.5 A ̊ and apparent sedimentation coefficients of 4.2 ± 0.2 s. Larger molecular species were also noted.

Analysis of the Humoral Immune Response to Influenza Virus in Vitro

The induction of in vitro primary and secondary humoral immune responses to influenza virus in murine splenic explant culture is described. Anti-influenza antibody synthesized in vitro was detected and quantitated by a radioimmunoassay which utilized influenza coupled to bromoacetylcellulose. Both in vitro primary and secondary responses could be stimulated over a large range of virus doses. In vitro secondary responses were maximal when spleen donors had been immunized by the parenteral route although secondary type responses could be demonstrated as well after primary immunization by pulmonary infection. In vitro stimulation with influenza virus was relatively insensitive to inhibition at high antigen doses. The results are discussed in terms of the response of other antigens in spleen fragment culture.

Antibody response by cultured spleen fragments from carrier-primed mice to hapten-protein conjugates.

Hapten-protein conjugates stimulated very poor anti-hapten responses in mouse spleen fragment cultures from unimmunized mice, whereas hapten coupled to type III pneumococcal polysaccharide or polylysine induced good responses. When the donors of the fragments were primed with the carrier protein, hapten-protein conjugates induced a strong anti-hapten response. Both the true primary and the carrier-primed response in vitro consisted mainly of IgA antibodies of 9-13S. In carrier-primed responses also IgM was produced at the beginning and IgG at the end of those responses.

The long-term organ culture of tissues from adult Necturus maculosus, the mud puppy.

Fragments of Necturus maculosus liver, spleen and kidney were cultured at 25°C in 50% Minimal Essential Medium (MEM) or 50% Leibovitz L-15 Medium (L-15) for up to 49 days. The integrity of tissue structure was evaluated, hepatocyte cell and nuclear volumes were measured, the respiration rates of freshly-isolated and cultured liver fragments were determined, and the mitotic incidences in cultured liver, spleen and kidney were estimated. The addition of adrenalin caused a reduction in the glycogen content of liver cultures, and the subsequent addition of insulin resulted in a net increase in glycogen synthesis. Glycogen levels fell in fragments cultured in L-15, but rose in cultures in MEM. Arginase and ornithine transcarbamylase levels fell gradually throughout a 49-day culture period in L-15. Evidence presented supports the position that the survival of tissues in vitro is related to cell size and respiration rate. These experiments show that N. maculosus is a suitable donor of tissues for long-term organ culture studies on the maintenance and control of tissue-type specific structure and function.

Graft-versus-host reaction in F 1 recipients in the absence of donor (parental) cell proliferation.

AbstractThis study concerns the role of donor cell proliferation in graft‐versus‐host (GVH) reaction, using in vivo and in vitro systems. We have confirmed the reports of others that treatment of parental cells with mitotic inhibitors prevents the development of GVH splenomegaly in newborn F1 recipients. However, in an in vitro GVH assay, parental cells treated with mitomycin C at doses as high as 50 μg/ml were shown to cause enlargement of newborn F1 spleen fragments. This apparent anomaly was partly resolved by showing that mitomycin C‐treated parental cells could give rise to splenomegaly in newborn F1 mice in vivo, provided they were mixed with newborn F1 spleen cells prior to injection. The possibility that mitotic inhibitors adversely affect the ability of parental cells to migrate to the sites of interaction with recipient cells, rather than inhibiting their actual ability to interact (and hence cause proliferation) is discussed. The nature of this GVH interaction is also considered.

Effect of erythropoietin on the spleen of the polycythemic mouse in vitro: V. Localization of erythropoietin-responsive cells in the spleen

The localization of erythropoietin-responsive cells (ERCs) in the mouse spleen was investigated in vitro. A thin fibrous spleen ‘capsule’ obtained by squashing the organ against a steel mesh was cultured with erythropoietin. The heme synthesis rate in the capsule was compared with that in the spleen fragments from the inner or the outer part of the organ. A far greater heme synthesis was found in the ‘capsule’ than in the other parts of the spleen. On the contrary, the heme synthesis rate in the spleen from anemic mice was higher in the outer part than in the capsule. It is suggested that a larger number of ERCs are located in the ‘capsular’ fragments than in the other portions of the organ. The ERCs may have affinity for the fibrous capsular structure when they are in the undifferentiated stage. The capsular tissue may provide a favorable microenvironment for the differentiation of the ERC.

Oligomeric IgA: the major component of the in vitro primary response of mouse spleen fragments.

The primary antibody response elicited from mouse spleen explants by conjugates of the 3-nitro-5-iodo-4-hydroxyphenylacetic acid (NIP) hapten consisted mostly of the IgA class. Poly-L-lysine, pneumococcal polysaccharide Type SIII, keyhole limpet hemocyanin, and sheep erythrocytes were effective carriers in this system, whereas chicken globulin was not. The anti-NIP response against all of the immunogenic conjugates was detectable in culture media 4 days after explantation and immunization, and reached peak titers by 8-10 days. IgA was identified by sucrose gradient velocity centrifugation in conjunction with the use of a class-specific antiserum. The media collected at 4 days contained low titers of IgM antibody, whereas the peak response at 8 days consisted almost entirely of IgA. The primary response IgA secreted by the spleen fragments was characterized as polymeric by its sedimentation rate through a sucrose gradient, and as polyvalent by its drastically greater avidity for NIP(14)BSA than for free NIP-aminocaproic acid. Its haptenated phage-inactivating activity was abolished by treatment with 0.1 M 2-mercaptoethanol. These experiments indicate that precursor cells existing in the spleen before primary immunization can give rise to production of polymeric IgA.

Enumeration and Analysis of Antibody-Forming Cell Precursors in the Neonatal Mouse

Abstract The ability of antibody-forming cell precursors (B cells) obtained during murine development to participate in the splenic focus response to a hapten-protein conjugate was determined by the technique of adoptive cell transfer to irradiated, syngeneic, carrier-primed recipient mice. Spleen fragments from recipients of fetal liver, fetal spleen, or neonatal spleen cells produced anti-hapten antibody when exposed in in vitro primary organ culture to dinitrophenylated hemocyanin (DNP-Hy). The number of antibody-producing fragments (splenic foci) obtained was linearly related to the number of nucleated spleen cells injected, and did not vary significantly for neonatal spleen cell populations obtained during the 1st week of postnatal life. Although neonatal foci released less antibody than that reported for adult foci (1), the dose dependence of antigenic stimulation is comparable for both neonatal and adult primary precursor cells, implying comparable mechanisms for antigenic recognition and stimulation. The frequency of precursor cells in the neonatal spleen for the haptenic determinant DNP is only slightly lower than the reported frequency (1) of adult precursor cells of the same specificity. This finding is discussed in terms of the relevance of antigenic selection to the generation of diversity.

Antibody of restricted heterogeneity secreted by spleen fragments.

SMALL fragments of lymphoid tissue in culture will incorporate radioactive amino-acids into secreted immunoglobulin. Electrophoresis followed by autoradiography has been used to indicate the degree of heterogeneity of the immunoglobulin1–5. Fragments of lymph node from a rabbit immunized with low doses of Salmonella organisms2–4 and of spleens from reconstituted irradiated mice immunized with sheep erythrocytes5 have been studied.

The long-term organ culture of tissues from adult Amphiuma, the Congo eel.

ABSTRACT Fragments of pancreas, liver, spleen, kidney and lung from Amphiuma means, the Congo eel, were maintained in organ culture for up to 35 days in a modified Leibovitz L15 medium. These Amphiuma tissues retained their typical histological features throughout the culture period, surviving better and for longer than tissues from other amphibian species. Liver and spleen survived better in hypotonic or isotonic media (125–230 mosmol/kg) than in hypertonic media (255–305 mosmol/kg). The mitotic incidences of these tissues in vivo were very low, and while there were no significant increases in liver, spleen and lung fragments in vitro, there were large increases in kidney and pancreas fragments. Amylase activity was found in pancreas fragments and in medium from pancreas cultures after 28 days in vitro. Amphiuma organ cultures may be useful in the in vitro study of the control of cell proliferation and cell function while normal cell interactions are maintained.

The impact of different motor activity on body composition, density of capillaries and fibers in the heart and soleus muscles, and cell's migration in vitro in male rats.

Male rats were subdivided according to motor activity in subgroups running daily on a motor-driven tread-mill and hanging on vertical ladders during 4–6 hrs per day (A), further in subgroups limited arteficially in motor activity in small cages (L) from the 18th to 65th (I) and from 18th to 285th day of life (II), and compared with controls (C). Both in 65th and 285th days of life the animals with increased motor activity had significantly increased caloric input, lowest weight and fat proportions, relatively heaviest liver, adrenals, tibialis and soleus muscles per 100 g body weight. Lowest caloric input in limited animals was found. Highest weights and fat proportions in control animals were found, with average caloric input. There existed no delayed consequences of changed motor regime from 18th to 65th days of life which were studied after period of latency at the age of 285 days (III). — The density of capillaries and muscle fibers per mm2 showed only significantly greater density of capillaries in soleus muscle in animals with increased motor activity at the age of 65 days (I), but not at the age of 285 days (II). There were no differences in capillary and muscle fiber densities as well as further indicators (capillary: fiber ratio, diffusion distance D/2) in the heart muscle in any of groups. Also the migration activity of cells from liver and spleen fragments in vitro studied in 285-days old animals (II) did not show differences as regards motor activity; neither there existed any prolongation of migration activity of cells from the heart and skeletal muscle fragments in vitro at this age.

Surface immunoglobulins on bursa-derived chicken lymphoid cells.

Cell suspensions from the spleen of normal and hormonally-bursectomized chickens were reacted with <sup>125</sup>I-labelled anti-immunoglobulin proteins of different class specificity. Following autoradiography, it was demonstrated that 22.5%, 24.3% and 11.8% of normal spleen cells labelled with radioiodinated anti-light chain, anti-<i>μ</i> chain, and anti-<i>γ</i> chain, respectively. The efficacy of bursectomy was confirmed by the absence of <i>in vitro</i> immunoglobulin synthesis by spleen fragments. No labelling was observed with any anti-immunoglobulin protein on spleen cells from bursectomized birds. Since maternal serum immunoglobulins were still present in the bursectomized birds, the possibility of cytophilic antibody being a cause of normal cell labelling was remote. The results support the hypothesis that the bursa-derived (B) cell is the equivalent of the mammalian nonthymus-derived (B) lymphocyte since both have a high surface density of immunoglobulin.

Induction of Immunity and Tolerance to the Dinitrophenyl Determinant in vitro

THE immune response in dissociated lymphoid cell cultures offers an opportunity to investigate the interaction of antigen with the surface receptors of immunocompetent cells. Using polymerized flagellin of Salmonella adelaide (POL), evidence was obtained that in vitro processes as different as immunity and tolerance both depend on the direct interaction between antigen and antigen-sensitive cells1–4. The use of chemically defined determinants in place of natural antigens could simplify the study of the molecular mechanisms underlying immunity and tolerance. Systems used in the past to induce immunity to defined determinants in vitro involved either a particulate antigen5 or spleen fragment cultures6 and were therefore unsuitable for the detailed study of the interactions occurring on the surface of lymphoid cells. A new system had to be devised. Here I describe the induction of a primary immune response to a hapten–protein conjugate in dissociated spleen cell cultures and the immune tolerance to a chemically defined determinant in vitro.

Cellular Aspects of Primary Antibody Response in Vitro

Abstract A membrane filter, modified organ culture system has been used in order to study the interrelationships between different cell types during the induction of a primary hemolytic response against sheep erythrocytes. Under these conditions, the normal architecture of the spleen fragments is preserved and a multi-cell transfer leading to antibody synthesis can be readily analyzed. The kinetics of the in vitro response using pre-immunized fragments were studied and compared with the kinetics of the in vivo response which it resembles. The kinetics of the primary response induced in vitro were also followed and analyzed with respect to the participation of the cells. The cells sort themselves into different populations on the filter.

Synthesis of the Second Component of Guinea Pig Complement In Vitro

Abstract Synthesis of hemolytically active C2 was detected in short-term tissue cultures of guinea pig spleen and to a lesser extent in cultures of lung, lymph nodes and cells obtained from bone marrow and peritoneal exudates. The in vitro production of C2 by these tissues was highly temperature-dependent and was reversibly inhibited by puromycin and cycloheximide. Fragments of spleen incorporated 14C-amino acids into a molecule that was immunochemically and functionally identical with C2. In vitro production of C2 was not detected in short-term cultures of kidney, skin, muscle, intestinal tract and liver.

The Induction of Bacteriophage-Neutralizing Antibody by Mouse Spleen Immunogenic RNA

Abstract RNA extracted from the spleens of Swiss Webster mice immunized with bacteriophage 81a, which infects Streptococcus faecalis var. zymogenes, induced the formation of specific phage-neutralizing antibody when added to nonimmune mouse spleen fragments in culture. Antibody formation could be elicited by the addition of immunogenic RNA derived from one inbred mouse strain to nonimmune spleen fragments from other inbred mouse strains. The activity of immunogenic RNA was inhibited by treatment with RNase, but not by DNase, trypsin, amylase or moderate amounts of pronase. The active RNA sedimented in the lightest fraction of a 5% to 20% linear sucrose gradient, and was estimated to have an average sedimentation coefficient of 8.2 S by analytical ultracentrifugation.

The Induction of Bacteriophage-Neutralizing Antibody by Mouse Spleen Immunogenic RNA

Abstract Antigenic components derived from phage were detected in immunogenic RNA preparations extracted from immune mouse spleens. Injection of immunogenic RNA into previously immunized mice resulted in an anamnestic response, whereas challenge with non-immune RNA did not. Treatment of immunogenic RNA with mouse antiphage antibody reduced its ability to induce neutralizing antibody in cultures of normal spleen fragments. The extracted RNA was found to contain 14C-labeled material derived from phage. These findings indicate the presence of antigen in RNA extracted from the spleens of immunized mice.

Transmission of an amphibian lymphosarcoma to and through insects.

Lymphosarcoma-bearing spleen fragments or cell-free extracts from an amphibian (<i>Xenopus laevis laevis</i>) implanted or injected into cockroaches (<i>Periplaneta americana</i>) elicited a characteristic hemocytic reaction. Back-transfer of the abnormal insect tissue to immature <i>Xenopus</i> up to 125 days after implantation led to the development of lymphoid tumors in the toads, thus indicating retention of infectivity during passage through the insect.