bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypocomplementemia in Feline Lymphosarcoma
Authors: Kobilinsky L. , Hardy W. D. , Kassel R. , Old L. J. , Day N. K.
Published: January 2023

The feline leukemia virus (FeLV) is a contagiously spread oncornavirus that causes malignant transformation of lymphoid cells. Feline cells transformed by FeLV produce a new cell surface antigen, the Feline Oncornavirus Associated Cell Membrane Antigen (FOCMA), which is a tumor-specific cellular antigen specified by, but not part of, the virion (Hardy, in press). An indirect immunofluorescent antibody test for FeLV antigens in leukocytes of peripheral blood indicates that approximately 90% of cats with lymphosarcoma (LSA) are infected with FeLV. In response to infection the cat may produce FeLV neutralizing and/or FOCMA antibodies.

In the present study functional levels of total complement (TCH50), C4, C2, and Factor B were determined in cat sera. Protein levels of C1q were estimated by radial immunodiffusion using monospecific antiserum against cat C1q. A total of 60 animals were studied, 32 of which were normal and FeLV negative.
Isolated Deficiencies of the Fifth and Eighth Components of Complement (C) in two Families: Clinical, Genetic and Biological Correlations
Authors: Snyderman R. , Pike M. C. , McCarty G. A. , Ward F. E.
Published: January 2023

Rapid advances in our understanding of the biochemistry and biology of C are being made, but the precise role of C in vivo is still poorly defined. The correlation of isolated deficiencies of C components in humans with clinical status, inheritance, and biological activity in vitro will better elucidate the function of C in vivo. In the past year we have identified two patients with isolated C component deficiencies. A severe deficiency of C5 (<2% of normal) was found in a previously healthy 21-year-old women with disseminated gonococcemia. Antibodies to DNA or to nucleoprotein were undetectable. In one year, despite vigorous antibiotic therapy and counseling, she had five recurrences of gonococcemia. Her twin sister (C5 <2% of normal) had one episode of suspected gonococcemia but has been otherwise healthy. Neither of the twins had any C5 protein detectable by radial immunodiffusion. The mother, a brother, and his son had C5 levels of ca. half normal.
Autoimmune Response to DNA, Red Blood Cells, and Thymocyte Antigens in Association with Polyclonal Antibody Synthesis During Experimental African Trypanosomiasis
Authors: Kobayakawa Takatoshi , Louis Jacques , Izui Shozo , Lambert Paul-Henri
Published: January 2023

In vivo induction of autoantibodies by polyclonal B cell activators has been suggested to be the direct consequence of the polyclonal B lymphocyte activation. Since the appearance of various autoantibodies represents a common feature of some parasitic infections, the possibility that such autoantibodies develop as a result of the stimulation of polyclonal antibody synthesis was tested in mice infected experimentally with Trypanosoma brucei brucei (T. brucei).

Polyclonal antibody production in the spleen was measured by the Cunningham plaque-forming cell (PFC) assay with SRBC, TNP-SRBC, and FITC-SRBC as indicator cells. Compared with normal mice, T. brucei-infected animals displayed a marked increase in the number of PFC to TNP, FITC, or SRBC. The level of polyclonal antibody synthesis was correlated to the number of T. brucei injected and to the number of parasites in circulating blood. The observed B lymphocyte activation is not dependent upon the influence of T cells since infection of athymic nu/nu mice also led to polyclonal antibody synthesis. Stimulation of polyclonal antibody synthesis was also observed after T. brucei infection of C3H/HeJ mice, a strain of mice that is nonresponsive to bacterial lipopolysaccharides. Therefore, it is unlikely that the lipopolysaccharides component of endogenous Gram-negative bacteria play an important role in the observed proliferation and differentiation of B lymphocytes.

Autoimmune responses to DNA, red blood cells, and thymocyte antigens were observed in mice infected with T. brucei and the kinetics of their formation was parallel to the development of the polyclonal B cell activation.
Quantitative Measurement of C9 Sites and Their Association to the Ring-Like “Lesions” on Complement-Lysed Membranes: A Morphometric Immunoferritin Study
Authors: Rauterberg Ernst W. , Ungemach Bäurbel , Gebest Hans-Joachim
Published: January 2023

The appearance of ring-like “lesions” on negatively stained target membranes that had been subjected to complement (C) lysis was observed in the electron microscope more than a decade ago. However, it remained unclear whether the ring-like structures represent micelles or bubbles in the outer leaflet of the membrane or the lytic C5-9 complex itself. In the present work by immunoelectronmicroscopy and morphometric studies the ring-like structures were identified to contain C9, and thus seen to be identical to the proposed C5-9 complex.

EAC 1–9 membranes were incubated with rabbit anti-C9 antibodies followed by ferritin-labeled goat anti-rabbit IgG antibodies. More than 50% of the ring-like structures could be labeled by the staining system. The association of C9 to the C rings was statistically significant (p < 0.001) as measured by two independent morphometric methods. About 50% of the C9-representing label could be observed with a greater distance than 300 Å apart from the C-induced ring-like structures. This finding may suggest that some of the ring-like structures are hidden on the membrane by their unfavorable position or that the ferritin label visualizes C9 not associated with ring-like structures. A possible diversity of the C5-9 complexes is discussed.
Physical Association of Histocompatibility Antigens and Tumor-Associated Antigens on the Surface of Murine Lymphoma Cells
Authors: Callahan Gerald N. , Allison James P. , Pellegrino Michele A. , Reisfeld Ralph A.
Published: January 2023

Serologic and immunochemical techniques were used to characterize plasma membrane antigens of the murine lymphoma 6C3HED. Syngeneic C3H/HeJ anti-6C3HED antisera were prepared and were shown by cytotoxicity, absorption, and immunoprecipitation tests to be specifically reactive with the lymphoma cells. Alloantisera to H-2.23 were found to be unreactive with intact lymphoma cells by cytotoxicity and absorption assays. However, H-2.23 antigens were detectable in tumor cell lysates by inhibition of cytotoxicity and immunoprecipitation. SDS-PAGE electropherograms of immunoprecipitates of radiolabeled tumor cell extracts obtained with anti-6C3HED and anti-H-2.23 antisera were essentially identical; proteins with Mr of approximately 70,000, 45,000, and 12,000 were apparent in both. An immunoadsorbent of insolubilized anti-6C3HED removed H-2.23 antigens detectable in lymphoma extracts, but had no effect on H-2.23 antigens in normal splenocyte extracts. These data indicate that H-2.23 and TAA are physically associated on 6C3HED cells.
Lymphocyte Stimulation with Fc Fragments
Authors: Berman Monique A. , Spiegelberg Hans L. , Weigle William O.
Published: January 2023

The effect of Fab and Fc fragments of immunoglobulins (Ig) of different species and different classes on DNA synthesis of mouse spleen cell cultures was analyzed by measuring 3H-thymidine incorporation. The data show that Fc fragments from mouse IgG induce strong stimulation of DNA synthesis in mouse spleen cells. In contrast, Fc fragments from turkey IgG are not stimulatory. The Fc fragments of all four human IgG subclasses, IgM, IgA1, and IgD, but not IgE, were stimulatory. In contrast, intact Ig and Fab fragments of the same classes had no effect.

Reduction and alkylation of the IgG Fc fragment did not diminish its activity. The pepsin fragment of IgG, pFc', which represents largely the Cγ3 domain, as well as a fragment obtained by trypsin cleavage of Fc also representing the Cγ3 domain, were both able to induce DNA synthesis. However, the activity of these subfragments was only between 10 and 25% of the activity of intact Fc, suggesting that sites in addition to the Cγ3 domain are required for optimal cell activation.

We postulate that enzymatic cleavage of Ig produces a mitogenically active Fc fragment with reactive sites similar to those of the Fc portion of Ig in antigen-antibody complexes or aggregated Ig. DNA synthesis induced by Fc fragments may represent a model of polyclonal lymphocyte activation through the Fc receptor.
Lactoperoxidase Catalyzed Radioiodination of Cell Surface Immunoglobulin: Incorporated Radioactivity May Not Reflect Relative Cell Surface Ig Density
Authors: Wilder Ronald L. , Chew Yuen Catherine , Mage Rose G.
Published: January 2023

Rabbit and mouse splenic lymphocytes were radioiodinated by the lactoperoxidase technique, extracted with non-ionic detergent, immunoprecipitated with high titered rabbit anti-kappa antisera, and compared by SDS-PAGE. Mouse sIg peaks were reproducibly larger in size than rabbit sIg peaks (often greater than 10 times). Neither differences in incorporation of label into the rabbit cell surface, nor differences in average sIg density explain this result. Total TCA-precipitable radioactivity was similar in each species. Estimation of the relative amounts of sIg in the mouse and rabbit showed similar average sIg densities. Differences in detergent solubility, proteolytic lability, or antisera used also do not adequately account for this difference. Thus, these data indicate that radioactivity incorporated after lactoperoxidase catalyzed cell surface radioiodination may not reflect cell surface Ig density. Conclusions about cell surface density based upon relative incorporation of radioactivity should be confirmed by other approaches.
Antigen-Specific Desensitization of Human Basophils
Authors: Sobotka Anne K. , Dembo Micah , Goldstein Byron , Lichtenstein Lawrence M.
Published: January 2023

Previous studies with isolated cell systems have shown desensitization (i.e., the induction of immunologic unresponsiveness after exposure to a stimulus under suboptimal conditions) to be antigenically nonspecific. Using pollen antigens and a bivalent penicillin antigen, [BPO]2, we found that, under conditions of passive sensitization, antigen-specific basophil desensitization occurred. Nonspecific desensitization always occurs with anti-IgE, and basophils of most atopic donors show nonspecific desensitization to antigens. Atopic donors were found, however, whose cells could be desensitized to one antigen while responding to noncross-reacting antigens and anti-IgE. Kinetic studies of desensitization revealed no differences between cells undergoing specific vs nonspecific desensitization. In the [BPO]2 system, removal of the antigen from the basophils by washing or by hapten ([BPO]1) competition did not reverse the specific desensitization to [BPO]2; passive replacement of specific IgE antibody did restore the response. Two types of desensitization, therefore, exist: 1) specific, which probably occurs due to the functional loss of some part of the IgE antibody-receptor complex and 2) nonspecific, involving the change noted above as well as an intracellular, biochemical alteration. Our data indicate that basophils with few IgE molecules undergo specific desensitization, whereas nonspecific desensitization occurs in basophils with many surface IgE molecules.
Binding of Antigen-Bearing Fluorescent Liposomes to the Murine Myeloma Tumor Mopc 315
Authors: Leserman Lee D. , Weinstein John N. , Blumenthal Robert , Sharrow Susan O. , Terry William D.
Published: January 2023

Small unilamellar lipid vesicles bearing the DNP-hapten on their surfaces and containing the water-soluble fluorescent dye carboxyfluorescein were formed by sonication. These vesicles were incubated with cells from the murine myeloma tumor MOPC 315, which secrete and also bear on the cell surface an immunoglobulin with affinity for the nitrophenyl hapten. At 0°C the cells bound an average of several thousand vesicles at saturation. This binding was specific for the nitrophenyl hapten on the vesicle since it was abolished by an excess of soluble nitrophenyl derivative, by omission of the hapten from the vesicle, or by substitution for MOPC 315 of a tumor lacking receptors for the nitrophenyl hapten. Specific binding of vesicles was greater when cells were incubated at 37°C.

The study suggests that ligand-bearing vesicles can be a useful marker for cell surface immunoglobulin. However, in spite of the ability to “target” vesicles to cell surface determinants, binding did not result in increased delivery of vesicle contents to the cytoplasm.
Reduced in Vitro Production of Anti-Tetanus Toxoid Antibody after Repeated in Vivo Immunization with Tetanus Toxoid
Authors: Stevens Ronald H. , Saxon Andrew
Published: January 2023

Lymphocytes from individuals booster-immunized with soluble tetanus toxoid synthesized anti-tetanus toxoid antibodies in vitro in the presence of pokeweed mitogen. The in vitro responsiveness initiated by day 14, peaked between days 20 and 40 and declined by days 60 to 80. Although the temporal kinetics of in vitro antibody production was similar in all individuals, the maximum levels of synthesis differed over an 8-fold range. The maximum quantity of anti-tetanus toxoid antibody synthesized in vitro after experimental immunization was correlated with the interval from prior booster immunization. Thus, cultures of B (0.4 × 106) and T (1.6 × 106) lymphocytes from individuals receiving the first immunization in ≥48 months synthesized up to 170 units of anti-tetanus toxoid antibody in vitro whereas individuals reboosted within 4 months of a prior immunization synthesized less than 40 units. This correlation was not observed with the in vitro synthesis of total IgG.

The in vitro anti-tetanus toxoid antibody response appeared to reflect in vivo production of antibody in that individuals receiving the first immunization in ≥48 months showed the greatest increase in serum antibody levels whereas a second booster immunization 4 months later caused only a slight increase in serum levels of anti-tetanus toxoid antibody. The decreased in vitro responsiveness after repeated immunization was not correlated with the suppressive activity of radiosensitive T lymphocytes but appeared to reflect decreased function of the non-T lymphocyte fraction. Removal of phagocytic cells did not increase in vitro anti-tetanus toxoid antibody production by lymphocytes from reboosted individuals.
Fc Receptors on Human T-Lymphocytes
Authors: Shaw Stephen , Pichler Werner J. , Nelson David L.
Published: January 2023

After in vitro allosensitization, human peripheral blood lymphocytes mediate specific allogeneic cell-mediated lysis (CML) and antibody-dependent cellular cytotoxicity (ADCC). To analyze the T lymphocyte subpopulations involved in CML and ADCC, lymphocytes were separated on the basis of their affinity for sheep erythrocytes (T cells), for Fc-IgM (TM cells), and Fc-IgG (TG cells) either before or after allosensitization. Subpopulations of T cells highly enriched for TM cells generated CML comparable to unseparated T cells, but subpopulations enriched for TG and effectively depleted of TM cells did not generate strong CML. Studies of CML generation by T cells exposed to immune complexes and by cocultured TM and TG cells failed to demonstrate any suppression by TG cells. Separation of subpopulations after allosensitization revealed that CML activity was mediated by T cells that lacked detectable Fc-IgG or Fc-IgM receptors. Parallel studies of ADCC revealed that the cytotoxic activity was depleted by removal of TG cells either before or after allosensitization. These studies demonstrate that a) TM cells contain precursors of CML effectors; b) TG cells are not required for the generation of CML; c) TG cells do not have marked suppressor activity in CML generation; d) TG cells contain ADCC effectors; and e) TM cells are not required for ADCC.
In Vitro Sensitization of Human Lymphocytes Against Histiocytic Lymphoma Cell Lines
Authors: Treves Abraham J. , Heidelberger Ellen , Kaplan Henry S.
Published: January 2023

Normal PBL were sensitized in vitro against an allogeneic diffuse histiocytic lymphoma cell line and their activity was measured by radiolabel release from target cells. We have reported earlier that a non-T cell population, found among the PBL, was responsible for inhibiting in vitro sensitization. In the present work we found that culturing PBL in vitro caused the induction of radioresistant suppressor cells which affected the sensitization phase of the in vitro response. The culture-induced suppressor cells had macrophage-like characteristics. The activity of the suppressor cells depended on an additional helper population that was adherent to nylon wool, but did not adhere to plastic and was nonphagocytic. The cooperation of these two different cell populations was required for the expression of suppressive activity.
The Late Phase Skin Reaction: Evidence for Activation of the Coagulation System in an IgE-Dependent Reaction in Man
Authors: deShazo Richard D. , Levinson Arnold I. , Dvorak Harold F. , Davis Robert W.
Published: January 2023

The late phase reaction (LPR), an IgE-dependent sequel to the wheal and flare response (WFR), was studied in ragweed (RW)-sensitive subjects 15 min and 6 to 8 hr after intradermal RW injection. The role of WFR in the genesis of LPR and LPR dependence on RW dose were determined by skin test titration to extinction of LPR. LPR size correlated with WFR size. LPR were noted with WFR as small as 9.5 ± 1.0 mm (S.E.M.) and RW doses as small as 0.01 protein nitrogen unit (PNU). In a group of WFR-positive but LPR-negative subjects, LPR were not induced despite increasing WFR to 18.2 ± 1.3 mm by simultaneous injection of RW + histamine (RW-H). Light microscopy of 1-µm biopsy sections of direct and Prausnitz-Kustner (P-K) LPR revealed a mixed cellular infiltrate qualitatively no different than that seen 6 hr after isolated WFR. Fibrin deposition in the absence of immunoglobulin or complement was seen by fluorescent microscopy in LPR but not in controls.

These data suggest that LPR may follow small RW doses. Furthermore, the failure of RW-H injection to induce LPR argues against a gatekeeper role for WFR in the genesis of LPR. The significance of the cellular infiltrate is not clear although fibrin deposition explains the clinical induration of LPR. This fibrin deposition is evidence for activation of the coagulation system in these IgE-dependent “delayed in time” reactions.
Studies of Congenitally Immunologic Mutant New Zealand Mice
Authors: Gershwin M. Eric , Castles James J. , Ikeda Richard M. , Erickson Kent , Montero John
Published: January 2023

Hereditarily asplenic (Dh/+) New Zealand Black (NZB) mice were produced by initial mating with B6CBA Dh/+ mice, followed by backcrossing, including H-2 typing and parental selection, with a colony of NZB mice. NZB Dh/+ mice, in their F6, F7, and F8 generation, were serially followed and compared to +/+ littermates. With age, NZB Dh/+ and +/+ mice develop a similar frequency of anti-erythrocyte and anti-nuclear, SS DNA, native DNA and poly I·poly C antibodies. Furthermore, renal histology is unchanged in NZB Dh/+ compared to +/+ mice. However, in striking contrast, NZB Dh/+ mice have a significantly reduced degree of hypergammaglobulinemia, a delayed onset and lower titer of naturally occurring thymocytotoxic antibody (NTA), and a reduction in both diffuse lymphocytic infiltrates and lymphoma. Moreover, although NZB Dh/+ mice lose a significant degree of T helper function with age, as manifest by reduced responsiveness to T cell mitogens, SRBC, and skin allografts, they appear to retain antigen nonspecific suppressor function. Thus, NZB Dh/+ mice develop autoimmune disease without influence by NTA and without premature loss of suppressor cell function. These results suggest that several of our present concepts regarding NTA, autoimmunity, and lymphoma in NZB mice need re-examination. Indeed, it appears that several features of NZB immunopathology, including development of NTA and lymphoma, are not directly related to the primary appearance of autoimmune disease.
Inhibition of Human Killer Cells by Autologous Lymphocytes
Authors: Pollack Sylvia B. , Emmons Sandra L.
Published: January 2023

Human antibody-dependent cellular cytotoxicity (ADCC) mediated by K cells is shown in this study to be inhibited by autologous lymphocytes. Inhibitor activity resides in a population of lymphocytes lacking Fc receptors, i.e., depletion of Fc receptor-bearing lymphocytes on immobolized enriches for inhibition. A T cell-enriched population does not inhibit. The effect is not steric inhibition since addition of large numbers of sheep or chicken erythrocytes does not decrease ADCC. Spontaneous cytotoxicity mediated by NK cells in the absence of added antibody is not inhibited by the FcR-depleted population, indicating that K and NK cells differ from each other in this respect.
Anti-tumor effects of allogeneic bone marrow transplantation in (NZB X NZW)F1 hybrids with spontaneous lymphosarcoma.
Authors: Moscovitch M , Slavin S
Published: January 2023

Untreated female (NZB X NZW)F1 hybrid mice (B/W F1) were found to develop lymphosarcoma spontaneously as they aged. Tumor incidence was evaluated in B/W F1 mice immunosuppressed with total lymphoid irradiation (TLI) and in TLI-conditioned B/W F1 mice reconstituted with 3 X 10(7) BALB/c bone marrow (BM) cells. BALB/C leads to B/W F1 chimerism (79 to 89% BALB/c-type cells) was confirmed by typing peripheral blood lymphocytes with specific alloantisera and complement by using a microcytotoxicity assay. Chimeras showed no clinical signs of graft-vs-host disease (GVHD). TLI-treated mice seemed to show a slightly accelerated onset of lymphosarcoma as compared with untreated controls, but the difference was not significant (p = 0.08). BALB/c leads to B/W F1 chimeras reconstituted at 1 to 3 mo of age (25 mice) developed no tumors for an observation period of 18 mo after transplantation. In contrast, tumors developed in 24/130 of age-matched controls, and in 13/57 of TLI-treated nonreconstituted age-matched B/W F1 mice. Tumor incidence in BALB/c leads to B/W F1 chimeras transplanted at an older age (9 to 11 mo) was similar to that observed in age-matched TLI-treated B/W F1 mice and age-matched untreated controls. The data suggests that the high naturally occurring incidence of lymphosarcoma could be reversed by reconstituting TLI-treated mice with BM cells (p = 0.027). Thus, allogeneic BM transplantation may exert potent graft-vs-tumor effects (GVT) when tumor susceptible hosts are reconstituted at an early age, whereas GVT is relatively ineffective at an advanced age, which probably correlates with an advanced stage of tumor development. Allogeneic BM transplantation should be additionally explored as a potential clinical tool for eradication of certain solid tumors in adjunct to high-dose radiochemotherapy, inasmuch as GVT seems to be independent of GVHD.
Thy-1 antigen expression by murine high-proliferative capacity hematopoietic progenitor cells. I. Relation between sensitivity to depletion by Thy-1 antibody and stem cell generation potential.
Authors: Boswell H S , Wade P M , Quesenberry P J
Published: January 2023

Hematopoietic stem cells of high proliferative potential such as the giant macrophage colony-forming cell HPP-CFC, were present in the marrow of mice treated with high dose 5-fluorouracil (5Fu) (150 mg/kg i.v.), whereas most committed granulocyte-macrophage progenitors, GM-CFU-C, were depleted. Enrichment of primitive stem cells in post 5-Fu bone marrow (5FuBM) was reflected in an enhanced capacity to proliferate in suspension cultures stimulated by the mixture of lymphokines present in Con A spleen-conditioned medium supernatant (Con A CM) when compared to normal bone marrow. The population of blast-like cells harvested at 5 days from suspension cultures of 5FuBM with Con A CM showed marked increases in stem cells GM-CFU-C and HPP-CFC. For this reason, 5FuBM was utilized to study the cell surface characteristics of putative pluripotential stem cells capable of giving rise to committed stem cells in suspension cultures. Treatment of 5FuBM (BDF1 mice) before suspension culture with a high concentration of either of two cytotoxic monoclonal antibodies directed against the Thy-1.2 surface antigen in the presence of rabbit complement reduced or abrogated the generation of stem cells HPP-CFC and GM-CFU-C in suspension cultures, even though the input content of HPP-CFC and GM-CFU-C in treated 5FuBM compared with control 5FuBM showed little reduction by the antibody plus complement treatment. The Thy-1+ cell required for generation of stem cells was not a T cell, because reconstitution of Thy-1.2-depleted 5FuBM with spleen nylon nonadherent (T) cells did not reconstitute the generation of stem cells, even though T cells did grow in the suspension cultures. In addition, depletion from 5FuBM of cells expressing Lyt-1 and Lyt-2 antigens, unambiguous markers of T cell-thymocyte differentiation, did not ablate the generation of HPP-CFC and GM-CFU-C. Rather, performance of Thy-1 cell depletion at lower efficiency, which still abrogated T cell function, ablated generation of HPP-CFC but did not affect the generation of GM-CFU-C. It was concluded that 5FuBM contains distinct Thy-1+ primitive stem cells expressing different amounts of Thy-1 antigen correlating with their respective generation potentials. Some of these Thy-1+ progenitor cells may be pluripotential.
B cell lines as models for inherited phagocytic diseases: abnormal superoxide generation in chronic granulomatous disease and giant granules in Chediak-Higashi syndrome.
Authors: Volkman D J , Buescher E S , Gallin J I , Fauci A S
Published: January 2023

Epstein Barr virus (EBV)-transformed B cell lines (BCL) were developed from peripheral blood lymphocytes of individuals with Chediak-Higashi syndrome (CHS) and chronic granulomatous disease (CGD), and were compared to EBV BCL from normals. The cells that were studied expressed both B cell surface antigens and EBV nuclear antigens. BCL from normals contained small cytoplasmic granules. When stimulated with phorbol myristate acetate (PMA), these B cells reduced nitroblue tetrazolium (NBT) and generated significant amounts of superoxide. However, two other phagocytic stimuli, A23187 and f-methionine-leucine-phenylalanine, failed to stimulate BCL oxygen metabolism. BCL from seven individuals with CGD failed to either reduce NBT or generate superoxide on PMA stimulation, although the lines were morphologically similar to BCL from normals. In contrast, CHS-BCL generated superoxide more rapidly than did normals, and contained the characteristic giant granules of CHS. Thus, continuous BCL evidencing the phenotypic abnormalities of inherited human phagocytic diseases can provide large numbers of cells for biochemical and genetic study.
Oligosaccharide structure of human C4.
Authors: Chan A C , Atkinson J P
Published: January 2023

The oligosaccharide structure of human C4 was studied by using C4 purified from plasma and C4 secreted by human hepatoma-derived cell line, HepG2. The alpha- and beta-chains of human C4 are glycosylated, whereas the gamma-chain is devoid of carbohydrate. The alpha-chain has three complex fucosylated oligosaccharides of the biantennary type, one each on the alpha 2, alpha 3, and alpha 4 fragments. The beta-chain has a single high mannose oligosaccharide primarily of the Man9GlcNAc2 type. The approximately 2000 Mr difference between the alpha-chains of the two C4 gene products (C4A and C4B) was localized to the alpha 2 fragment and is not due to carbohydrate. Sulfation of the C4 alpha-chain was localized to the alpha 4 fragment of the alpha-chain. Hence, the Mr difference between the two gene products is likely to reside in amino acid differences. The oligosaccharide structure of three incompletely processed C4 molecules was also analyzed. These molecules have the oligosaccharide composition of the appropriate individual subunits. Therefore, intracellular proteolytic processing to the multi-chain form of C4 is not required for proper oligosaccharide processing.
Human endothelial cells modulate granulocyte adherence and chemotaxis.
Authors: Zimmerman G A , Wiseman G A , Hill H R
Published: January 2023

Although human endothelial cells (EC) and granulocytes interact in several ways, the factors that regulate such interactions are not well defined. In this study we found that EC and their products directly altered granulocyte adherence (GA) and chemotactic activity. The spontaneous adherence of granulocytes to human umbilical vein EC monolayers was significantly reduced at 5, 15 and 30 min if the granulocytes were preincubated with EC that were stimulated by rocking. At 30 min the spontaneous adherence of EC-preincubated granulocytes was 36% of that of control granulocytes (P = 0.004). The augmented adherence stimulated by FMLP was also decreased (54% of control) by preincubation of the granulocytes with rocked EC. The ability of stimulated EC to inhibit GA was attenuated when the EC monolayers that were used for preincubation with the granulocytes were pretreated with the cyclooxygenase inhibitor indomethacin. GA to unstimulated EC was not significantly altered by indomethacin or aspirin, suggesting that cyclooxygenase products do not influence GA under resting conditions. Preincubation of granulocytes with rocked EC monolayers or supernatant media from rocked EC monolayers diminished their chemotactic response to FMLP by 45 to 65. This inhibition was also attenuated by pretreatment of the EC with indomethacin. EC supernatant medium caused a rapid increase in granulocyte intracellular cyclic AMP, with a maximum increase to 200% of control at 1 min. These data indicate that stimulated EC release one or more arachidonic acid products that alter spontaneous and inflammatory mediator-stimulated granulocyte activity. Prostacyclin, a major cyclooxygenase product of EC arachidonate, depressed inflammatory mediator-augmented GA to EC monolayers and chemotaxis when present in nanomolar concentrations. We conclude that EC-derived prostacyclin, alone or in combination with other EC products, alters GA and chemotaxis stimulated by inflammatory mediators. This provides a mechanism by which EC may modulate granulocyte distribution as well as granulocyte responses that are influenced by adherence, such as the release of toxic oxygen metabolites and granular enzymes.
Murine TH response to influenza virus: recognition of hemagglutinin, neuraminidase, matrix, and nucleoproteins.
Authors: Hurwitz J L , Hackett C J , McAndrew E C , Gerhard W
Published: January 2023

BALB/c mice were primed with type A influenza virus by footpad injection or by aerosol infection with PR8 [A/PR/8/34-(H1N1)]. Isolated T cells from draining lymph nodes were then tested for their proliferation in the presence of purified viral proteins hemagglutinin, neuraminidase, matrix, and nucleoprotein. Significant responses [( 3H]thymidine incorporation) were seen against each of the four proteins after either priming scheme. When helper T (TH) cell clones were isolated by hybridoma formation from two different strains of mice, responsiveness (interleukin 2 production) towards each protein was against apparent. Of 12 virus-specific T cell hybridomas isolated, four responded to matrix, three to nucleoprotein, one to neuraminidase, three to hemagglutinin, and one cell was of undefined specificity. Each hybridoma was also tested for recognition of the HK virus [A/Hong Kong/1/68-(H3N2)], which differs in subtype from the priming strain. All matrix-specific cells, two nucleoprotein-specific cells, and the cell of undefined specificity were cross-reactive with HK virus. H1-subtype specificity was seen for all hemagglutinin and neuraminidase-specific cells and one of the three nucleoprotein-specific cells. Because many virus-immune TH cells recognize antigenically variable determinants, a significant fraction of TH cell function may be lost after virus evolution. When selecting priming schemes for long-term immunization against influenza, the isolated enhancement of TH cells recognizing conserved determinants on matrix and nucleoprotein may therefore be considered.
Interleukin 2 activates a receptor-associated protein kinase.
Authors: Benedict S H , Mills G B , Gelfand E W
Published: January 2023

The interleukin 2 (IL 2) receptor complex has been shown to consist of at least two IL 2 binding molecules, one of 55 to 57 kd (gp57Tac) and one of 75 to 78 kd apparent m.w. The data presented here indicate that a protein of m.w. 78,000 (pp78) co-immunoprecipitates with gp57Tac when a monoclonal antibody against gp57Tac is used. The 78 kd molecule is phosphorylated in vitro within the immune complex only in the presence of exogenously added IL 2, whereas the 57 kd molecule is phosphorylated equally in the presence or absence of IL 2. Phosphorylation in vitro of pp78 was demonstrated in extracts of human peripheral blood T cells (PBL-T) and the human T cell line Jurkat, but not in extracts of the human macrophage line U937 or the murine T cell line 2.8.2. Metabolic phosphorylation in intact cells reflects results observed in vitro; both pp78 and gp57Tac are phosphorylated in PBL-T and Jurkat, but not in U937. These data demonstrate that the IL 2 receptor complex contains an IL 2 responsive protein kinase activity and may signal the cell through a phosphorylation event.
The IRG-47 gene is IFN-gamma induced in B cells and encodes a protein with GTP-binding motifs.
Authors: Gilly M , Wall R
Published: January 2023

The murine pre-B cell line 70Z/3 has been studied extensively as a model system for B cell differentiation induced by cytokines and mitogens. IFN-gamma treatment of 70Z/3 pre-B cells activates Ig kappa L chain gene expression, resulting in the appearance of surface IgM characteristic of B cells. We have isolated and characterized a new cDNA clone, called IRG-47, whose mRNA is transiently elevated up to 30-fold at 4 to 6 h after IFN-gamma induction of 70Z/3 pre-B cells. The IFN-gamma induction of IRG-47 mRNA occurs in all pre-B and in most B lineage cell lines examined but is only induced in three cell lines tested from other lineages. Activation of IRG-47 gene transcription appears to account for the rapid increase in IRG-47 mRNA after induction. Transcription of the IRG-47 gene is down-regulated 10 h postinduction at a time when the level of IRG-47 mRNA is also declining. The complete sequence of the IRG-47 cDNA clone is predicted to encode a 47-KDa protein which, although unique in its sequence, bears resemblance to a number of nucleotide-binding proteins. The IRG-47 coding sequence contains all three highly conserved peptide motifs in the appropriate spacing characteristic of guanine nucleotide-binding proteins.
Tolerance induction in T helper (Th1) cells by thymic macrophages.
Authors: Jayaraman S , Luo Y , Dorf M E
Published: January 2023

Most macrophages in the peripheral tissues present Ag optimally to a variety of functionally distinct Th cells. Although thymic macrophages have been implicated in deleting autoreactive thymocytes, their role in influencing the functional capacities of mature T cells is not clear. We have established a normal untransformed macrophage cell line, named TMC, from the mouse thymus. The TMC line presents protein Ag to an IL-4-producing Th2 type Th clone after IFN-gamma treatment as evidence by T cell proliferation and the release of IL-3 and IL-4. However, these thymic macrophages are inefficient at stimulating a well characterized cytochrome C-specific IL-2-producing Th1 clone, A.E7. Ag presentation by TMC results in the production of IL-3 but not IL-2 production or proliferation of A.E7 cells. This selective Ag presentation defect to Th1 cells is corrected by the addition of live but not fixed allogeneic irradiated spleen cells, suggesting that the thymic macrophages lack the expression of costimulatory activity required for Th1 activation. This is further demonstrated by the failure of live thymic macrophages to provide costimulatory activity to A.E7 cells stimulated with fixed spleen cells plus the antigenic peptide 81-104. Exposure of A.E7 cells to paraformaldehyde-treated TMC in the presence of 81-104 peptide induces specific hyporesponsiveness, anergy. These data demonstrate that thymic macrophages can have a profound influence on the response of selected T cells to Ag. Furthermore, the nature of the T cell stimulus is also critical because Th1 and Th2 cells responded equally well to the T cell mitogen, Con A, and a bacterial superantigen presented by the thymic macrophages.
Mutations in the third, but not the first or second, hypervariable regions of DR(beta 1*0101) eliminate DR1-restricted recognition of a pertussis toxin peptide.
Authors: Olson R R , De Magistris M T , Di Tommaso A , Karr R W
Published: January 2023

We have examined the role of 12 polymorphic residues of the beta-chain of the HLA-DR1 class II molecule in T cell recognition of an epitope of pertussis toxin. Murine L cell transfectants expressing wild-type or mutant DR1 molecules (containing single amino acid substitutions in DR(beta 1*0101)) were used as APC in proliferation assays involving nine DR1-restricted T cell clones specific for peptide 30-42 of pertussis toxin. Four different patterns of recognition of the mutants were found among the pertussis-specific clones. Residues in the third hypervariable region (HVR) of DR(beta 1*0101) are critically important for all the T cell clones; amino acid substitutions at positions 70 and 74 abrogated recognition by all of the T cell clones, and substitutions at positions 67 and 71 eliminated recognition by most of the clones. In contrast, most single amino acid substitutions in the first and second HVR, predicted to be located in the floor of the peptide binding groove, had little or no effect on the proliferative responses of these clones. However, the involvement of beta-chain first and second HVR residues was demonstrated by the inability of transfectants expressing wild-type DR(beta 1*0404) (DR4Dw14) or DR(beta 1*1402) (DR6Dw16) to present peptide to these clones. These beta-chains have completely different first and second HVR compared with DR(alpha,beta 1*0101) although the third HVR are identical. These results illustrate the functional importance of third HVR residues of DR(beta 1*0101) and allow definition of the molecular interactions of the DR1 molecule with the 30-42 peptide.
Use of human universally antigenic tetanus toxin T cell epitopes as carriers for human vaccination.
Authors: Valmori D , Pessi A , Bianchi E , Corradin G
Published: January 2023

Synthetic constructs were assembled as multiple Ag peptide systems containing repetitive sequences of Plasmodium falciparum and Plasmodium berghei, the causative agents of human and murine malaria respectively, and two universal human tetanus toxin T cell epitopes 830-843 and 947-967. These constructs were tested for antibody production in mice and for their capacity to stimulate human PBL and tetanus toxin-specific T cell clones. A high antibody titer can be obtained in mice when multiple Ag peptide systems are injected in various adjuvants or in PBS alone. Furthermore, all constructs can activate PBL from every donor tested. However, a variable response was obtained when different clones specific for the two tetanus toxin universal epitopes were used. These constructs may represent possible candidates for a malaria vaccine.
T cell-induced allotypic suppression. Origin of the CD8+ T cells maintaining IgG2ab suppression.
Authors: Majlessi L , Benaroch P , Bordenave G
Published: January 2023

One of the problems raised by the T cell-induced allotypic suppression is the origin (donor or host) of the T cells responsible for the chronicity of the suppression. To address this point, we used T cells from Igha/a Thy-1.2 mice whose natural T cell activity against IgG2ab was enhanced in vivo. These T cells were injected into newborn Ighb/b Thy-1.1 mice where they induced complete suppression of IgG2ab expression in around 70% of these recipients. During a study that lasted more than 1 yr, we found that about 3% of the recipient splenocytes were T cells of the donor type. By means of suppression-transfer experiments, using either Thy-1.2+ or Thy-1.1+ cell-depleted splenocytes from mice suppressed in this manner we were able to unambiguously show that Thy-1.2+ cell-depleted splenocytes were incapable of transferring the suppression, whereas Thy-1.1+ cell-depleted splenocytes could. We thus demonstrated that suppression was maintained throughout the recipient's life by donor Thy-1.2+ T cells.
The beta 2-microglobulin dissociation rate is an accurate measure of the stability of MHC class I heterotrimers and depends on which peptide is bound.
Authors: Parker K C , DiBrino M , Hull L , Coligan J E
Published: January 2023

Stable, recombinant, water-soluble complexes of HLA-A2 and HLA-B27 were reconstituted from 125I-labeled beta 2-microglobulin (beta 2m), a synthetic peptide, and HLA H chain fragments expressed as inclusion bodies in the Escherichia coli cytoplasm. Using this system, we were able to show: 1) the t1/2 of beta 2m dissociation from HLA complexes at 37 degrees C varied from approximately 40 h to less than 1 h, depending on the peptide employed for reconstitution. Peptide length and composition were found to be critical factors in determining the beta 2m dissociation rate. Endogenous peptides form complexes that are about as stable as those formed with typical antigenic peptides. 2) Peptide exchange reactions, in which an exogenous peptide replaces the peptide that is already bound by the class I molecule, proceed readily for complexes that have rapid beta 2m dissociation rates. Thus, difficulties in demonstrating peptide binding to complexes that contain endogenous peptides can be attributed to the stability of the endogenous peptide/class I molecule complex. 3) The peptide exchange reaction does not require concomitant beta 2m dissociation. 4) Distal parts of the class I molecule, which are not directly involved in peptide binding or beta 2m binding, have a major impact on the stability of class I molecules. Thus, these studies show that the dissociation rate of beta 2m is an excellent measure of how tightly a given peptide binds to class I MHC molecules, that the ability to bind peptide is tightly coupled to the binding of beta 2m and vice versa, and that regions of the molecule distal from the binding site influence the stability of peptide binding.
Peptide binding to soluble HLA-DR4 molecules produced by insect cells.
Authors: Scheirle A , Takacs B , Kremer L , Marin F , Sinigaglia F
Published: January 2023

HLA-DR4Dw4 molecules were expressed in insect Sf9 cells. The transmembrane and cytoplasmic domains of the DR4 alpha- and beta-chains were replaced by the carboxy terminal sequence of decay accelerating factor, leading to a phosphatidyl inositol glycan membrane anchor. This structure contains a cleavage site for phosphatidyl inositol-specific phospholipase C, allowing efficient solubilization of the rDR4 molecules. We present evidence that infected insect cells express properly associated surface heterodimers and are able to present antigenic peptides to DR4Dw4-restricted T cell clones. Phosphatidyl inositol-specific phospholipase-cleaved recombinant molecules exhibited in vitro binding characteristics similar to DR4 molecules purified from lymphoblastoid cells. In terms of peptide specificity, pH optimum, kinetics, and affinity they were indistinguishable within the limits of our assay system. However, the peptide binding capacity of the recombinant molecules was higher than that of native DR4 molecules.
Analysis of HIV-induced autoantibodies to cryptic epitopes on human CD4.
Authors: Callahan L N , Roderiquez G , Mallinson M , Norcross M A
Published: January 2023

Antilymphocyte antibodies, including autoantibodies to CD4, have been reported in AIDS patients and are postulated to contribute to T cell depletion and immunologic dysfunction. In this paper, we characterize and localize binding sites of human anti-CD4 autoantibodies from a number of HIV+ patients. Epitope mapping by ELISA and Western blotting, together with cross-competition experiments, showed that common autoepitopes were localized to at least two topographically separate sites on the fourth domain of sCD4. These sites were partially dependent on the carboxyl terminus of the soluble molecule and were not exposed on full length membrane CD4, even under denaturing Western blotting conditions. Peptide screening identified peptides from the fourth and third domains that were recognized by several, but not all, anti-CD4 serum samples. Soluble CD4 affinity-purified antibodies were predominantly IgG1 and were not induced to bind mCD4 after gp120 binding to T cells. Analysis of HIV seroconversion panels showed that the appearance of anti-CD4 antibodies followed HIV seroconversion by 6 to 12 months and paralleled anti-gp120 reactivity. This suggested a correlation between immune reactivity to envelope and anti-CD4 antibody production. Together, the data indicate that human anti-CD4 antibodies recognize cryptic conformational and linear epitopes on a cleaved form of CD4. These findings suggest that HIV may induce abnormal cleavage of full length CD4, thereby exposing immunogenic self epitopes normally hidden from humoral and cellular immune interactions. This model of abnormal processing of self Ag has general implications for autoantigen exposure in other autoimmune disorders.
The Use of Trypan Blue to Demonstrate the Immediate Skin Reaction in Rabbits and Guinea Pigs
Authors: Ramsdell Susan Griffith
Published: January 2023

Summary and Conclusions

By the intravenous administration of trypan blue, the immediate skin reaction to a homologous antigen may be demonstrated in serum treated rabbits and guinea pigs in the usual testing for direct, passive and reversed passive sensitization in both animals.

The same type of reaction may be shown to an injection of sera of sensitized animals, of sera showing so-called primary toxicity for the guinea pig, and to very high dilutions of histamine.

It is believed that the infiltration of the dye into these reaction sites is, at present, to be looked upon as no more than a sort of indicator of the presence of an edematous change which is an expression of a toxic action upon the capillary endothelium.
Ultra Microscopic Findings in the Serum of the Anaphylactic Guinea Pig
Authors: Ramsdell Susan Griffith , Kast Clara C.
Published: January 2023

Using the ultra-microscope in a study of the serum of the sensitized guinea pig, we have found the flocculation phenomenon, as postulated by Kopaczewski, to occur irregularly, to appear to be simply a very weak precipitin reaction, and to have no constant relation to the capacity of the animal to show shock.

Certain changes in the appearance of the sera were often noted following shock, but the nature of these were not resolvable by the use of the technic as outlined.