bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and physicochemical characterization of murine T cell replacing factor (TRF).
Authors: Takatsu K , Harada N , Hara Y , Takahama Y , Yamada G , Dobashi K , Hamaoka T
Published: December 2022

Murine T cell replacing factor (TRF) was purified from a cellfree supernatant of a T cell hybridoma (B151K12) that constitutively produces TRF. Two assay systems for TRF activity were employed: 1) induction of anti-DNP IgG PFC responses in cultures of splenic B cells from DNP-KLH-primed BALB/c mice, and 2) induction of IgM PFC in chronic B cell leukemic cells (BCL1). The purification scheme consisted of ammonium sulfate precipitation, DEAE-cellulose chromatography, Blue-Sepharose chromatography, hydroxylapatite chromatography, gel permeation with fast protein liquid chromatography (FPLC), and disc polyacrylamide gel electrophoresis. Overall, TRF was purified approximately 34,000-fold with a maximum 3.8% recovery of activity, and the specific activity of the purified TRF was approximately 9.6 X 10(4) U/mg. The TRF that is active in these systems is distinct from the other lymphokines such as IL 1, IL 2, BCGFI (now known as BSFp1), and gamma-interferon. The TRF is extremely hydrophobic, with an apparent m.w. of 50,000 to 60,000 on gel permeation chromatography and 18,000 on SDS-PAGE under reducing conditions. Highly purified B151-TRF abrogated the activity by treatment with trypsin but not with RNase. Moreover, it bound to lima bean agglutinin-Sepharose specific for N-acetylgalactosamine residues, indicating that B151-TRF is a glycosylated glycoprotein containing N-acetylgalactosamine residues. The role of N-acetylgalactosamine residues on TRF activity was additionally substantiated by the fact that the addition of appropriate amounts of N-acetylgalactosamine in the assay systems for TRF preferentially induced a profound suppression for TRF-mediated PFC responses.
Demonstration of structural polymorphism among MB3 light chains by two-dimensional gel electrophoresis.
Authors: Ishikawa N , Kasahara M , Ikeda H , Ogasawara K , Hawkin S , Takenouchi T , Wakisaka A , Kikuchi Y , Aizawa M
Published: December 2022

The heavy and light chain subunits of MB3 molecules were isolated from KT2 (DKT2, DR4, MB3 homozygous), ER (Dw4, DR4, MB3 homozygous), JMe (Dw5, DR5, MB3 homozygous), EBV-Sh (DSh, DRw6.2, MB3 homozygous), and EBV-Ky (DKy, DRw9, MB3 homozygous) cells and were compared with one another by two-dimensional gel electrophoresis. The MB3 light chains from KT2, ER, and EBV-Ky cells were clearly different in terms of their isoelectric points, whereas those from ER, JMe, and EBV-Sh cells were indistinguishable. No differences in charge or m.w. were noted for the MB3 heavy chains from the five cell lines. Thus, three out of the five MB3-positive, D/DR-disparate cell lines were found to express structurally distinct MB3 molecules, demonstrating that MB3 is a public serologic specificity shared by at least three structurally distinct MB (human I-A-like) molecules. Because the DR light chain subunits isolated from EBV-Wa, KT2, ER, JMe, EBV-Sh, and EBV-Ky cells differed from one another in their isoelectric points, the DR light chains were apparently more polymorphic than the MB3 light chains.
Enhanced reactive lysis of paroxysmal nocturnal hemoglobinuria erythrocytes by C5b-9 does not involve increased C7 binding or cell-bound C3b.
Authors: Rosenfeld S I , Jenkins D E , Leddy J P
Published: December 2022

The most complement (C)-sensitive type of erythrocytes (E) occurring in paroxysmal nocturnal hemoglobinuria (type III PNH E) have previously been found to exhibit approximately twofold to fourfold greater lysis than normal human E when exposed to isolated human C5b6, C7, C8, and C9 (reactive lysis), in the absence of a known source of C3- or C5-convertases or fluid-phase C3. In further studies on the mechanism of this phenomenon, we now report that C5b6-dependent binding of 125I-C7 to two samples of PNH E (greater than 95% type III) is equal to that found with normal human E at each of several C5b6 inputs tested. Lysis developed by excess C8 and C9, however, was consistently greater for the PNH E. Thus, the exaggerated sensitivity of type III PNH E to reactive lysis cannot be explained by abnormally high uptake of C5b6 or C7 from the fluid phase. Rather, the data indicate that cell-bound C5b67 sites are converted to effective hemolytic sites with greater efficiency on type III PNH E than on normal human E, assuming that the distribution of cell-bound C7 throughout both cell populations is similar. In related studies we have addressed the proposal by other investigators that C3b putatively bound to PNH E in vivo might account for their increased sensitivity to reactive lysis in vitro, by analogy to prior observations on C3b-potentiated reactive lysis of sheep E. The latter hypothesis was made more appealing by the recent discovery that type III PNH E lack an integral membrane protein, decay-accelerating factor (DAF), which in normal E accelerates the decay of membrane-bound C3 convertases. Against this hypothesis, however, is our present finding that preincubation of PNH E with four different goat or rabbit polyclonal antibodies to human C3 failed to inhibit the subsequent reactive lysis of these cells. Under these same conditions, the C3b-dependent increment in reactive lysis of sheep EAC4b3b was abrogated by pretreatment with similar dilutions of these anti-C3 antibodies, generally in association with agglutination. Furthermore, sheep EAC4b3b displayed increased 125I-C7 binding in proportion to augmented lysis, in contrast to the findings with PNH E. Therefore, deficiency of DAF in type III PNH E does not adequately explain their supranormal sensitivity to reactive lysis unless DAF can modulate the terminal lytic steps by a mechanism distinct from its effect on C3 convertase decay. Alternatively, type III PNH E could have a more general abnormality in which DAF deficiency is one manifestation and increased sensitivity to reactive lysis is another.
Normal function of CR1 on affected erythrocytes of patients with paroxysmal nocturnal hemoglobinuria.
Authors: Roberts W N , Wilson J G , Wong W , Jenkins D E , Fearon D T , Austen K F , Nicholson-Weller A
Published: December 2022

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia in which affected erythrocytes (E) are abnormally sensitive to lysis by autologous complement. Affected E from patients with PNH (PNH-E) are deficient in an E membrane regulatory protein of complement, decay-accelerating factor (DAF). Because a functional defect in a second membrane regulatory protein of complement, CR1 (C3b receptor), has also been hypothesized, severely affected PNH-E (type III PNH-E) were tested for abnormalities in CR1 by four methods. E from two patients with 100% type III PNH-E had 3201 and 6783 sites per cell for binding of 125I-labeled rabbit polyclonal F(ab')2 anti-CR1. These values fall within the normal range of CR1 antigenic sites per cell (1267 to 7915, mean = 5,014 +/- 155 SEM) established by assaying the E from 113 healthy donors. The Ka of CR1 on type III PNH-E for 125I-labeled C3b dimer was 2.06 X 10(7) M-1, and the Ka values for the binding of the same ligand to the E from two healthy individuals were 2.45 X 10(7) M-1 and 1.58 X 10(7) M-1. In an assay designed to measure the capacity of human E (Eh) to accelerate the decay of the classical C3 convertase deposited on 1 X 10(7) bystander sheep E (EAC1gp,4bh,2agp), the half-life (t 1/2) of this convertase was diminished from 18.1 min (range 15.2 to 22.9) to 8.1 min (range 7.4 to 8.5) by the addition of 1 X 10(7) normal Eh, to 6.2 min by 100% type III PNH-E, and to 7.5 min by Eh pretreated with an IgG fraction of human antiserum directed against the D antigen of the Rh system. In contrast, Eh (t 1/2 = 7.4) pretreated with a saturating dose of F(ab')2 anti-CR1, and CR1-deficient Eh (less than 10 CR1 molecules/E) from a patient with systemic lupus erythematosus, showed a loss of convertase decay-accelerating capacity to t 1/2 = 11.6 and t 1/2 = 12.4, respectively. Type III PNH-E pretreated with anti-CR1 demonstrated a total loss of their decay-accelerating capacity (t 1/2 = 19.9). In an assay of I cofactor activity, soluble C3b was rapidly converted to iC3b by purified I plus Eh or type III PNH-E, whereas CR1-deficient Eh exhibited less than 5% the I cofactor activity of normal Eh.(ABSTRACT TRUNCATED AT 400 WORDS)
Morphologic and immunologic characterization of human basophils developed in cultures of cord blood mononuclear cells.
Authors: Ishizaka T , Dvorak A M , Conrad D H , Niebyl J R , Marquette J P , Ishizaka K
Published: December 2022

Selective growth of human basophilic granulocytes was obtained in suspension cultures of mononuclear cells from umbilical cord blood. Approximately 50 to 80% of nonadherent cells recovered from 2- to 3-wk-old cultures contained metachromatic granules, and these cells were identified as human basophilic granulocytes by electron microscopy. Histamine content of cultured human basophils was comparable to that in peripheral blood basophils. Cultured basophils bear 2.7 to 3.7 X 10(5) IgE receptors per cell that bind both human IgE and rodent IgE with comparable affinity. Average equilibrium constants of the receptors for human IgE and mouse IgE were 2.56 +/- 0.88 X 10(9) M-1 and 1.85 +/- 0.86 X 10(9) M-1, respectively. The cell-surface component of the IgE receptors on cultured basophils has a m.w. of 64,000. Cultured basophils could be passively sensitized with human IgE and mouse IgE monoclonal antibody, and sensitized basophils released characteristic cytoplasmic granules and both histamine and arachidonate upon challenge with either anti-human IgE or antigen. Incubation of cultured basophils with ionophore A23187 or F-Met-Leu-Phe resulted in histamine release. However, compound 48/80 failed to induce histamine release from the cells.
Complement- and IgE-mediated release of histamine from basophils in vitro. V. Differential effects of drugs modulating arachidonic acid metabolism.
Authors: Farnam J , Grant J A , Lett-Brown M A , Hunt C , Thueson D O , Giclas P C
Published: December 2022

Basophil degranulation probably plays a significant role in the pathogenesis of different hypersensitivity reactions. These cells can be stimulated to secrete lysosomal histamine in vitro in response to various secretagogues. We compared the effects of drugs, modulating arachidonic acid (AA) metabolism, on histamine release (HR) from human basophils stimulated by anti-IgE antibody or C5a anaphylatoxin. Leukocytes from normal donors were preincubated with drug for 15 min at 22 degrees C, followed by the addition of C5a or anti-IgE for 30 min at 37 degrees C. Bromophenacyl bromide, an inhibitor of AA formation by phospholipase, blocked the effects of C5a and anti-IgE (greater than or equal to 3.3 X 10(-6) M, p less than 0.05 and p less than 0.01, respectively). 3-Amino-1-(3-trifluoromethylphenyl)-2-pyrazoline hydrochloride (BW755C, greater than or equal to 3.3 X 10(-5) M) and 5, 8, 11, 14-eicosatetraynoic acid (greater than or equal to 3.3 X 10(-4) M), known inhibitors of both cyclooxygenase (COX) and lipoxygenase (LPX) pathways of AA metabolism, blocked both C5a- and anti-IgE-induced HR (p less than 0.01). Nordihydroguaiaretic acid, an inhibitor of LPX, decreased HR induced by anti-IgE (greater than or equal to 3.3 X 10(-6) M, p less than 0.01) and allergens, but reduced C5a-initiated HR only at a higher concentration (greater than or equal to 7 X 10(-5) M, p less than 0.01). Indomethacin (INDO), an inhibitor of COX, significantly reduced HR caused by C5a (greater than or equal to 3.3 X 10(-8) M, p less than 0.01) and its degradation product C5adesArg, but had no effect or caused slight enhancement of HR initiated by anti-IgE. We confirmed that INDO augments allergen-induced HR. Our findings suggest that there are basic differences in the regulation of C5a- and IgE-mediated basophil degranulation.
Regulation of cell-mediated immunity in cryptococcosis. III. Characterization of second-order T suppressor cells (Ts2).
Authors: Murphy J W , Mosley R L
Published: December 2022

Frequently, patients with systemic cryptococcosis have depressed or absent in vivo or in vitro cell-mediated immune responses to cryptococcal antigen. In addition, these patients have relatively high levels of circulating cryptococcal antigen, which is generally considered indicative of a poor prognosis. Because acquired cell-mediated immunity is an important host defense mechanism in cryptococcosis, we have been studying the effects of cryptococcal antigen on such responses. Using a murine model, we have demonstrated that cryptococcal antigen given i.v. to CBA/J mice, to simulate antigen levels found in patients, can trigger the production of a series of suppressor cells that specifically inhibit anticryptococcal cell-mediated immune responses. Briefly, cryptococcal antigen induces afferent suppressor and/or suppressor inducer (Ts1) cells in the lymph nodes of mice, and the Ts1 cells, or a soluble factor derived therefrom (TsF1), stimulate the production of second-order or efferent suppressor (Ts2) cells. This report provides data that demonstrate that the Ts2 cells were induced in spleens from cyclophosphamide (Cy)-resistant precursors in response to cryptococcal Ts1 cells or TsF1 and that a proliferative process sensitive to Cy was not required for Ts2 induction. The Ts2 cells were detectable 6 days after Ts1 injection, were specific in suppressing the cryptococcal delayed-type hypersensitivity (DTH) response, were T cells with an Lyt-1-,2+,Ia+ (I-J+) phenotype, were effective only on the efferent limb of the cryptococcal DTH response, and mediated their activity via a soluble factor (TsF2). Thus far, the suppressive pathway induced by cryptococcal antigen has characteristics more like those of the suppressor circuits described for the DTH responses to the haptens azobenzenearsonate and 4-hydroxy-3-nitrophenyl acetyl than those of the modulating circuits induced by other antigens. We postulate that a suppressive circuit similar to the one we have defined in the mouse model is operating to suppress the in vivo and in vitro cell-mediated immune responses in cryptococcosis patients who have significant amounts of circulating cryptococcal antigen.
Characterization of monoclonal antibodies to Treponema pallidum.
Authors: Lukehart S A , Tam M R , Hom J , Baker-Zander S A , Holmes K K , Nowinski R C
Published: December 2022

Thirteen hybrid cell lines which produce mouse monoclonal antibodies to Treponema pallidum, the causative agent of syphilis, have been established. All of the monoclonal antibodies react with T. pallidum, Nichols strain, in ELISA and in immunofluorescence assays, but do not react with normal rabbit testicular tissue in the ELISA. Two of these antibodies were demonstrated to react with the nonpathogenic treponemes T. phagedenis, biotype Reiter, T. refringens (Noguchi strain), T. vincentii, and T. denticola (strains 11 and W), as well as with Borrelia recurrentis, Leptospira interrogans, serogroup Canicola, and the swine pathogen T. hyodysenteriae. The remaining 11 antibodies react with four recently isolated strains of T. pallidum, but with none of the related nonpathogens nor with Borrelia or Leptospira. Thus, our results to date indicate that these monoclonal antibodies may identify antigenic determinants that are specific either for T. pallidum alone or for those treponemes which are pathogenic for humans. The molecular specificities of six of the 13 antibodies were determined by Western blotting. We anticipate potential usefulness of these antibodies in the investigation of the antigenic structure of T. pallidum, the taxonomic study of the pathogenic and nonpathogenic treponemes, and in the diagnosis of syphilis.
B cell subsets in spleens of BALB/c mice: identification and isolation of MMTV-expressing and MMTV-responding subpopulations.
Authors: Lopez D M , Charyulu V , Paul R D
Published: December 2022

The DNA of BALB/c mice contains two genomic- and one subgenomic-size MMTV proviruses that appear to be preferentially expressed in their spleen cells, although intact MMTV virions cannot be detected in the tissues of these mice. This retrovirus antigen expression is restricted to a subpopulation of B lymphocytes, as determined by double label immunofluorescence studies. Nylon-adherent, SIg-positive spleen lymphocytes from BALB/c mice are capable of being stimulated by purified MMTV in lymphocyte transformation assays. Two possibilities were explored: the MMTV-positive cells are the responders to MMTV in the blastogenesis assay, or there exists two B lymphocyte subsets, one expressing the MMTV antigen(s) and the other responding to the virus. Depletion of MMTV-positive, nylon-adherent cells with anti-MMTV and complement resulted in no significant change in the blastogenesis to MMTV, indicating that the MMTV-negative lymphocytes are the responders in this reaction. These results were confirmed by positive selection experiments by using a fluorescence-activated cell sorter. Two populations of nylon-adherent cells, on the basis of the presence or absence of MMTV antigen in their surfaces, were obtained by a two-way sorting procedure and were used in lymphocyte transformation assays. MMTV-expressing lymphocytes were found to be nonresponsive to MMTV antigens, although high levels of [3H]thymidine incorporation were observed in the MMTV-negative, nylon-adherent cell cultures exposed to MMTV. These data indicate that in normal BALB/c mice, expression of endogeneous retrovirus genetic information is restricted to a nylon-adherent spleen cell subset that is different from the one responding in blastogenesis to the viral antigens.
Functional analysis of mononuclear cells infiltrating into tumors: differential cytotoxicity of mononuclear cells from tumors of immune and nonimmune rats.
Authors: Ibayashi Y , Uede T , Uede T , Kikuchi K
Published: December 2022

A million syngeneic gliosarcoma (T-9) cells injected subcutaneously are sufficient to kill Fisher rats within 2 mo. Fisher rats became resistant to T-9 cells by surgical removal of the implanted tumor and repeated immunization with MMC-treated T-9 cells. Histopathologic studies revealed massive accumulation of mononuclear cells in tumor tissues of immune rats that were rejecting syngeneic T-9 tumors, whereas very few mononuclear cells were found in tumor tissues of nonimmune rats. Cell populations infiltrating into tumor tissues were identified by immunohistochemical techniques. Mononuclear cells found within the regressing tumors of immune rats were identified mostly to be T cells, and two-thirds of these T cells were OX-8 positive. In contrast, mononuclear cells found within the growing tumors of nonimmune rats were identified to be mixtures of macrophages and T cells, and very few OX-8 positive cells were found. Mononuclear cells were isolated from implanted T-9 tumors to determine whether mononuclear cells lysed T-9 cells specifically. Significant tumoricidal activity was seen when mononuclear cells from tumors of immune rats were used, whereas no detectable tumoricidal activity was observed with mononuclear cells from tumors of nonimmune rats. Winn assays confirmed in vitro 51Cr release assays by showing that tumors were rejected only when T-9 cells were implanted into normal Fisher rats along with mononuclear cells from tumors of immune rats.
Monoclonal helper T cells induce B cell responses to T-independent antigens: antigen-specific T cells are directly stimulated by activated B cells in the absence of antigen.
Authors: DeKruyff R H , Clayberger C , Cantor H
Published: December 2022

Efficient B cell responses to most polysaccharide antigens such as TNP-PAA or TNP-Ficoll require factors produced by activated T cells. However, the mechanism of T cell activation during such responses has not been established, because these antigens do not activate T cells, either directly or in conjunction with I-A gene products. We used a panel of antigen-specific monoclonal helper T cells to study T cell activation during the course of such responses. We show that activated I-A-identical B cells directly stimulate these monoclonal T cells, and that this stimulation is in the absence of nominal antigen. The high frequency of inducer cells that are stimulated by activated B cells suggests a major biologic role for this novel pathway of T cell activation.
Development and distribution of B lineage cells in the domestic cat: analysis with monoclonal antibodies to cat mu-, gamma-, kappa-, and lambda-chains and heterologous anti-alpha antibodies.
Authors: Klotz F W , Gathings W E , Cooper M D
Published: December 2022

To trace the development and distribution of B lineage cells in the domestic cat (Felis catus), we have produced monoclonal antibodies against mu-, gamma-, kappa-, and lambda-chains of feline immunoglobulins (Ig). Goat antibodies against human mu-, alpha-, and lambda-chains, which are reactive with shared determinants on their feline counterparts, were used in conjunction with the panel of mouse monoclonal antibodies. Cytoplasmic mu+ pre-B cells and surface IgM+ B lymphocytes were observed in 42 day fetal liver in which pre-B cells were more abundant than IgM+ B cells. Pre-B cells also were found in bone marrow in young cats, and continued to be generated in the marrow throughout life. In the spleen, adult levels of B cells were attained by 12 wk of age, at which time the frequencies of surface IgM+, IgG+, and lambda+ cells were 49, 3, and 40%, respectively. The distributions of Ig isotypes also were determined among plasma cells as a function of age and tissue localization. IgM plasma cells were predominant in the bone marrow of 1-wk-old cats, whereas IgG plasma cells were the prevalent isotype in adult bone marrow. In the mesenteric lymph nodes of adult animals, the frequency distributions of IgM, IgG, and IgA plasma cells were similar to the frequency distributions of IgM, IgG, and IgA isotypes among bone marrow plasma cells. IgA+ plasma cells predominated in the intestinal lamina propria, in which IgG+ and IgM+ plasma cells were relatively infrequent. In the tissues of both young and adult animals, the ratio of lambda:kappa expression was approximately 3:1. We conclude that the pattern of B cell development in the cat resembles that found in other mammals, except that the kappa to lambda ratio is reversed.
Stimulated rat T cell-derived inhibitory factor for cellular DNA synthesis (STIF). II. Kinetics of the production and characterization of the producer.
Authors: Chiba K , Nishimura T , Hashimoto Y
Published: December 2022

Kinetics of the production of a stimulated T cell-derived inhibitory factor for cellular DNA synthesis (STIF) (1) from Con A-stimulated SD rat spleen cells, and the cells involved in the production of the factor, were examined comparatively with those of interleukin 2 (IL 2) and interferon (IFN). STIF activity in the culture supernatants reached a plateau 24 hr after the culture, and the plateau level was maintained during an additional culture for 72 hr. Characterization of the cells involved in STIF production by means of negative selection of unfractionated or nylon-fractionated spleen cells with anti-rat T cell serum or monoclonal antibodies plus complement revealed that the cells are nylon-nonadherent T cells bearing a suppressor T cell marker. The nylon-nonadherent T cell population did not require additional macrophages for STIF production. In vivo pretreatment of rats with cyclophosphamide (25 to 100 mg/kg) reduced or abolished the production of STIF from the Con A-stimulated spleen cells of the rats. The STIF production from Con A-stimulated spleen cells was inhibited by the addition of indomethacin at 10 ng/ml, indicating a regulatory role of prostaglandin(s) in STIF production. The above characteristics distinguish a T cell subset for STIF production from those for IL 2 and IFN production.
Biosynthesis of mouse Thy-1 antigen.
Authors: Luescher B , Bron C
Published: December 2022

The biosynthesis and the maturation of Thy-1 antigen of mouse thymocytes have been studied by using a xenogeneic rabbit anti-mouse Thy-1 antibody. The earliest form of Thy-1 detected after a 5-min pulse with [35S]methionine and [35S]cysteine had an apparent m.w. of 26,500. During chase, this band converted to a molecular ratio (Mr) = 25,000 polypeptide, probably derived from the latter by trimming of glucose or mannose residues from the three high-mannose glycan units of Thy-1. Mature Thy-1 molecules were detected at the cell surface after a 15-min chase. At least one of the three N-linked oligosaccharide units was shown to be in the high mannose form at the cell surface, as indicated by its susceptibility to endo-beta-N-acetylglucosaminidase H digestion. Treatment of the early and late forms of Thy-1 antigen with endo-beta-N-acetylglucosaminidase F generated a single polypeptide of Mr = 13,500. The same precursor was obtained when cells were labeled in the presence of tunicamycin. This indicates the absence of O-linked glycan in the mature cell surface antigen. Finally, the resistance of Thy-1 antigen to trypsin digestion when associated with membranes confirmed that this molecule has no cytoplasmically oriented portion.
Isolation of rabbit C3, Factor B, and Factor H and comparison of their properties with those of the human analog.
Authors: Horstmann R D , Müller-Eberhard H J
Published: December 2022

The rabbit complement components C3, Factor B, and Factor H were isolated and characterized and were compared to the corresponding proteins of human serum. Chromatographic behavior, chemical properties, and functional interactions show great similarities between the components in both species. By SDS polyacrylamide gel electrophoresis, the m.w. were estimated to be 195,000 for C3, 86,000 for Factor B, and 155,000 for Factor H. The amino acid compositions of the rabbit proteins resembled those of the human analog. The total carbohydrate content of rabbit C3 and Factor H was approximately one-half that of the human proteins. In addition, a qualitative difference in the carbohydrate moieties of the C3 proteins was observed. The serum concentration of the rabbit proteins was markedly lower than that of the human proteins. The rabbit C3b,Bb enzyme resembled the human analog with respect to half-life, control by Factor H, and stabilization by nickel ions.
Complement-mediated hemolytic activity of succinylated concanavalin A: preparation and activity of cell intermediates.
Authors: Langone J J , Ejzemberg R
Published: December 2022

Succinylated concanavalin A (SCon A) lyses sheep erythrocytes (E) in the presence of complement, whereas the native tetravalent lectin, Con A, is inactive. We have studied the ability of E-SCon A (ES) to interact with early acting guinea pig (gp) or human (hu) complement components (C1, C2, C4) and found that cell intermediates ESC1, ESC4, ESC14, and ESC142 can be generated that are analogous to intermediates conventionally prepared with E and rabbit IgM (pentameric) anti-Forssman antibody. Titration of gp or hu C1, C4, and C2, and quantification of the number of activated C1 molecules bound to ESC4 by the C1 fixation and transfer test showed in each case that an average of one effective site per cell was sufficient to cause cell lysis. Determination of tmax for optimal formation of ESC142 sites depended on the species combination of components used to make the intermediates, and the decay of ESC142 and EAC142 sites or sites generated with ESC4, EAC4, and trypsin-activated C2 were similar. The sugar alpha-D-methylglucopyranoside (alpha-MGP) inhibited binding of SCon A to E and eluted the lectin from ES, whereas galactose was nearly inactive, consistent with lectin sugar-binding selectivity. In contrast, both sugars were ineffective in eluting SCon A or C4hu from ESC4hu, indicating that C4hu blocked the interaction between lectin and alpha-MGP, perhaps by steric hindrance. SCon A is a divalent functional analogue of IgM anti-Forssman antibody that may be a uniquely suited reagent specific for cell membrane glycoconjugates for studying the mechanism of binding and activation of complement components.
Modulation of cytoplasmic calcium in human platelets by the phospholipid platelet-activating factor 1-O-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine.
Authors: Valone F H , Johnson B
Published: December 2022

The calcium-sensitive, fluorescent dye Quin 2 was used to quantitate changes in free intracellular calcium [( Ca2+]i) induced in platelets by the phospholipid platelet-activating factor 1-O-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine (AGEPC). The Ca2+]i of unstimulated platelets was 91 +/- 18 nM (mean +/- SD, n = 8), and treatment with 1 to 16 nM AGEPC increased [Ca2+]i in a dose-related manner, with 16 nM AGEPC increasing [Ca2+]i by 102 +/- 20 nM. [Ca2+]i was not increased by analogs of AGEPC which do not activate platelets including the lysophospholipid precursor of AGEPC, the optical isomer, and a C-2 benzoyl analog. The capacity of AGEPC to increase [Ca2+]i exceeded that required to induce maximal platelet aggregation. In four experiments, 100% platelet aggregation was induced by 4.5 +/- 2.4 nM AGEPC (mean +/- SD) and was associated with a submaximal increase in [Ca2+]i of 56 +/- 22 nM. Pretreatment of platelets with AGEPC rendered the platelets specifically unresponsive to repeat stimulation with AGEPC in terms of both platelet aggregation and increased [Ca2+]i, whereas the platelet response to thrombin was undiminished by pretreatment with AGEPC. In contrast, the platelet response to 0.5 microM calcium ionophore A23187 was undiminished by pretreatment with the same concentration of ionophore, suggesting that AGEPC does not activate platelets by an ionophore-like mechanism. IgG aggregates and AGEPC in combination activate platelets synergistically, as shown by the observation that a 1-min exposure of platelets to 60 micrograms/ml of IgG aggregates increased the platelet aggregation response to 2 nM AGEPC from 44 to 100%. In contrast, sequential exposure of platelets to IgG aggregates and AGEPC increased [Ca2+]i additively, suggesting that increased [Ca2+]i contributes to but does not fully mediate synergistic platelet activation by IgG aggregates and AGEPC. Quantitation of free intracellular calcium with the fluorescent dye Quin 2 is a highly sensitive technique for delineating the role of calcium in mediating platelet activation.
Effect of antibodies directed against complement receptors on phagocytosis by polymorphonuclear leukocytes: use of iodination as a convenient measure of phagocytosis.
Authors: Klebanoff S J , Beatty P G , Schreiber R D , Ochs H D , Waltersdorph A M
Published: December 2022

Two monoclonal antibodies (Mab), designated 60.3 and 60.1, markedly inhibited the phagocytosis of serum-opsonized zymosan by human polymorphonuclear leukocytes (PMN) as measured by the iodination reaction and by microscopic visualization. These antibodies also inhibited rosette formation with EC3bi without decreasing EC3b rosetting, suggesting that Mab 60.3 and 60.1 inhibit the phagocytosis of opsonized zymosan through reaction with the C3bi receptor (CR3) on the leukocyte surface. In support of this concept is the finding that the PMN of two patients with recurrent infections do not ingest opsonized zymosan, lack C3bi receptor function, and react weakly or not at all with Mab 60.3 and 60.1. At concentrations which completely inhibited ingestion of opsonized zymosan, both Mab partially inhibited iodination with Staphylococcus aureus 502A as the particle, and did not affect iodination when Staphylococcus epidermidis was used. This presumably reflects a variable need among the opsonized particles for CR3 for ingestion. Mab 60.3 also inhibited the phagocytosis of certain unopsonized particles as measured by iodination, indicating that the antigens recognized by the Mab do not influence phagocytosis solely by functioning as a C3bi receptor. Mab 60.3 increased the phagocytosis of unopsonized, heat-killed S. aureus by reaction with the PMN via its antibody-combining site, and with the staphylococcal protein A via its Fc region (reverse opsonization). This process required protein A-containing organisms (S. aureus 502A or Cowan 1 but not S. aureus Wood 46 or S. epidermidis), was inhibited by purified protein A, and was not seen either when the F(ab')2 or Fab fragments of the antibody, or when PMN which lack or have low levels of the antigen were employed. Thus, these studies, using iodination as a convenient method for the measurement of phagocytosis, demonstrated two effects of antibodies directed against PMN cell surface components: inhibition of phagocytosis by reaction with the C3bi receptor, and stimulation of phagocytosis by reverse opsonization.
Effect of glucocorticoids on the development of suppressive activity in human lymphocyte response to a polysaccharide purified from Candida albicans.
Authors: Piccolella E , Vismara D , Lombardi G , Guerritore D , Piantelli M , Ranelletti F O
Published: December 2022

The action of glucocorticoids on the proliferative response of human lymphocytes stimulated in vitro by MPPS has been investigated. The effect of Dex was dependent on the time of steroid addition to the cultures. Dex added at the beginning of the culture period inhibited, cell proliferation and IL 1/IL 2 synthesis, although not completely. However, a delayed addition of 24 to 48 hr resulted in an enhancing effect on cell proliferative responses that was maximal at day 4. The effect of Dex on T suppressor cell activity was then investigated. Dex added 1 day before the appearance of suppressor cells resulted in a marked decrease or disappearance of the suppressive activity. Moreover, primed T lymphocytes treated with Dex in the presence of exogenous IL 2 enhanced the proliferative responses of fresh autologous PBMC stimulated by MPPS. Taken together, our data suggest that glucocorticoids inhibit the differentiation of T suppressor cells and that IL 2 is unable to reverse this inhibitory effect.
Mouse IgD half molecules with shortened IgD heavy chain result from alterations within C delta locus.
Authors: Thiele C J , Owens J D , Finkelman F D , Mushinski J F
Published: December 2022

An unusually small (51 KD) IgD myeloma protein was isolated from secretions of TEPC 1017 generation (gen) 24. The delta-chain mRNA and the delta-chain gene in this tumor were compared with those of TEPC 1017 of earlier generations. The gen 24 protein contained one normal-sized kappa-type light chain (21 KD) and one unusually short delta-heavy chain (30 KD). The delta-heavy chain was 15 KD shorter than that of TEPC 1017 of earlier generations, owing to a delta-mRNA (1.15 kb) which was 600 bp shorter than that of TEPC 1017 of earlier generations. TEPC 1017 is a tetraploid tumor, and the gen 24 appears to contain at least two different deletions on different chromosomes. The short mRNA was produced from one of these altered delta-chain genes which had a productive VDJ rearrangement but which had lost the C delta 3 domain and perhaps the C delta H domain as well. Despite these genetic insults, RNA splicing produced delta-mRNA with secreted termini and mRNA with membrane-binding termini. It is suggested that the mouse C delta gene has an unusual predilection for deletions because it normally lacks any vestige of C delta 2 and, during i.p. passage, it suffered further deletions or alterations.
Lymphokine-activated tumor inhibition in vivo. I. The local administration of interleukin 2 triggers nonreactive lymphocytes from tumor-bearing mice to inhibit tumor growth.
Authors: Forni G , Giovarelli M , Santoni A
Published: December 2022

CE-2 is a chemically induced tumor of low immunogenicity in syngeneic BALB/c mice. Nylon wool columns eluting lymphocytes from the spleen of mice bearing clinically evident (5-mm mean diameter) CE-2 tumors (CE-2 TB lymphocytes) do not react with CE-2 cells in vitro, nor are they able to affect their growth in vivo in a Winn-type neutralization assay at 5:1 lymphocyte:tumor cell ratio. However, they become able to inhibit CE-2 tumor growth when 20 U of interleukin 2 (IL 2) in 0.4 ml are injected daily for 10 days at the challenge site. In contrast, mice injected with CE-2 cells and IL 2 only display tumor takes and growth that are not significantly different from those in controls challenged with CE-2 cells alone. This lymphokine-activated tumor inhibition (LATI) is not a peculiarity of the CE-2 tumor-host combination, because different tumors can be inhibited in this way and various TB lymphocytes can initiate it. In these experiments, IL 2-rich 25,000 to 30,000 m.w. fractions were obtained routinely from the culture supernatants of a clone of EL-4 thymoma stimulated with phorbol myristic acetate. Equally active IL 2-rich preparations were obtained from rat spleen cells stimulated with concanavalin A, or from MLA 144 gibbon lymphosarcoma spontaneously releasing IL 2. Treatment of CE-2 TB lymphocytes with various antibody and C, with 2000 rad gamma-irradiation, or fractionation on Percoll density gradients suggested that radioresistant functions of Thy-1.2+, Lyt-1.2+, Lyt-2.2- and of asialo GM1+ cells are independently involved in LATI induction. These lymphocytes inhibit tumor growth by recruiting the radiosensitive effector mechanisms of the recipient mice required for ultimate tumor destruction. CE-2 tumor inhibition by LATI leaves a specific delayed-type hypersensitivity and an immunologic memory, resulting in rejection of a second lethal CE-2 challenge in a significant number of mice.
T cell regulation of antibody responses: an I-A-specific, autoreactive T cell collaborates with antigen-specific helper T cells to promote IgG responses.
Authors: Clayberger C , DeKruyff R H , Cantor H
Published: December 2022

In earlier studies we showed that hapten-specific inducer T cell clones specifically induce B cells from immunized donors to secrete IgM antibodies. However, IgG responses were not observed, suggesting that an additional signal(s) was required. In this report, we show that an autoreactive T cell clone produces a factor(s) that collaborates with antigen-specific inducer T cells to promote specific IgG responses. This factor is not restricted by antigen or MHC determinants and promotes IgG production both in vivo and in vitro. These findings suggest that autoreactive cells may play an important role in the regulation of isotype expression.
Treatment of murine lupus with monoclonal anti-T cell antibody.
Authors: Wofsy D , Ledbetter J A , Hendler P L , Seaman W E
Published: December 2022

Three strains of autoimmune mice (MRL/lpr, NZB/NZW, and BXSB) were treated with repeated injections of rat monoclonal anti-T cell antibody (anti-Thy-1.2) in order to determine 1) the extent and duration of target cell depletion, 2) the effect of T cell depletion on the course of autoimmunity, and 3) the magnitude and consequences of the host immune response to the monoclonal antibody. Mice were treated with 6 mg of anti-Thy-1.2 every 2 wk beginning early in their disease. Treatment produced a substantial reduction in circulating T cells in all three strains. Therapy was beneficial in MRL/lpr mice. It reduced lymphadenopathy, lowered autoantibody concentrations, retarded renal disease, and prolonged life. In contrast, treatment did not improve autoimmunity in NZB/NZW mice, and it caused fatal anaphylaxis in BXSB mice. These findings demonstrate that monoclonal antilymphocyte antibodies can serve as specific probes to examine the cells that contribute to autoimmunity. Moreover, they illustrate the potential therapeutic value of monoclonal antilymphocyte antibodies when a pathogeneic cell subset can be identified. However, the same antibody may have a broad range of effects, from efficacy to severe toxicity, even in diseases that share clinical features.
Lupus prone (SWR x NZB)F1 mice produce potentially nephritogenic autoantibodies inherited from the normal SWR parent.
Authors: Gavalchin J , Nicklas J A , Eastcott J W , Madaio M P , Stollar B D , Schwartz R S , Datta S K
Published: December 2022

The incidence of nephritis in autoimmune NZB mice is low, but when they are crossed with normal SWR mice, almost 100% of the female F1 hybrids (SNF1) develop lethal glomerulonephritis. To define the contribution of the normal SWR strain to the development of nephritis, we analyzed 65 monoclonal anti-DNA autoantibodies derived from SNF1 mice and compared them with those obtained from the NZB parent. The majority of the SNF1-derived anti-DNA antibodies were IgG and cationic in charge. By contrast, 77% of the NZB-derived antibodies were IgM. Moreover, all three NZB-derived IgG anti-DNA antibodies were anionic. The cationic property of the SNF1-derived IgG autoantibodies was not restricted to any particular antigenic specificity pattern or IgG subclass, nor was there a preference for the allotype of either parent. However, we identified a set of highly cationic (pI at 8.2 to 8.8 pH) IgG2b anti-DNA antibodies from SNF1 hybrids that had the SWR allotype. Isoelectric focusing of intact antibodies and isolated heavy and light chains showed that the highly cationic charge of these antibodies was determined by the variable regions of their heavy chains. Because IgG anti-DNA antibodies with cationic charge are especially pathogenic, those antibodies bearing the allotype of the normal SWR parent may account for the high incidence of severe nephritis in the F1 hybrids. The results indicate that pathogenic autoantibodies, which are encoded by genes of the nonautoimmune SWR parent, are expressed in the SNF1 mice due to some cellular and genetic regulatory influence of the NZB parent.
A cloned MCGF cDNA encodes a multilineage hematopoietic growth factor: multiple activities of interleukin 3.
Authors: Rennick D M , Lee F D , Yokota T , Arai K I , Cantor H , Nabel G J
Published: December 2022

We recently isolated from a mouse T cell cDNA library a full-length clone that encodes a mast cell growth factor (1). On the basis of sequence homologies, this cloned factor must be similar if not identical to purified interleukin 3 (IL 3) (2, 3). Here, we report the first biologic characterization of the cloned gene product expressed in COS-7 monkey cells. Our results establish that a single molecular species promotes the growth and differentiation of a wide spectrum of hematopoietic cell types, including multipotential stem cells and various committed progenitor cells. The relationship of cloned IL 3 to other colony-stimulating factors is discussed.
Antigenic stimulation regulates the expression of IL 2 receptors in a cytolytic T lymphocyte clone.
Authors: Lowenthal J W , Tougne C , MacDonald H R , Smith K A , Nabholz M
Published: December 2022

We have isolated and characterized an H-2Kb-specific CTL clone, B1.8, whose growth is strictly dependent on IL 2 and periodic exposure to stimulator cells. A single, brief encounter with antigen induces on resting B1.8 cells the expression of high-affinity IL 2 receptors, which is maintained for several days, thus allowing cells to undergo at least six rounds of division in the presence of affinity-purified IL 2. We show with the use of monoclonal anti-Lyt-2 or -H-2Kb antibodies, which block specific antigen recognition by B1.8 cells, that continuous antigenic stimulation is not required during this period of growth. After this time, B1.8 cells lose their ability to respond to IL 2 and revert to a resting state, a process that is reversed upon addition of fresh stimulator cells. The decline and subsequent arrest of cell growth correlates with a loss of IL 2 receptors; growth-arrested cells express a 10-fold lower number of receptors than actively growing cells.
Isolated follicular dendritic cells: cytochemical antigen localization, Nomarski, SEM, and TEM morphology.
Authors: Szakal A K , Gieringer R L , Kosco M H , Tew J G
Published: December 2022

The objectives of the present study were to determine the cytological features of isolated follicular dendritic cells (FDC), which distinguish them from other leukocytes or dendritic cell types. Consequently, we have developed methods for the fixation, peroxidase cytochemistry, and visualization of FDC, which are applicable to cytological evaluations by Nomarski optics, scanning, and transmission electron microscopy. A functionally supported identification of FDC in vitro was made possible by utilizing, in conjunction with the dendritic morphology, the cytochemically identifiable antigen, horseradish peroxidase (HRP), and the known capacity of FDC to sequester immune complexes (i.e. HRP-anti-HRP) on their plasma membranes. The observations showed that FDC constitute a relatively pleomorphic, nonphagocytic group, distinct from other dendritic type cells such as lymphoid dendritic cells, Langerhans cells, and interdigitating cells (LDC, LC, and IDC), as well as typical leukocytes. Morphologically distinct FDC were identified as cells either with filiform dendrites or with "beaded" dendrites. FDC possessed a single or sometimes a double, lymphocyte-size cell body, which contained an irregular, lobated nucleus, Golgi apparatus, numerous small vesicles, and some mitochondria. Mitochondria were not abundant in the dendritic processes. Filiform dendrites tended to branch and anastomose near the cell body and form a radiating "sunburst"-like pattern. On the average, dendrites measured 15-20 microns in length and 0.1-0.3 micron in diameter. Occasional dendrites were extremely elongated, reached several hundred microns in length, and terminated in an enlargement measuring nearly a micron in diameter. Other filiform dendrites usually had a club-shaped terminal enlargement. The microspheres of "beaded" dendrites ranged between 0.3 and 0.6 micron in diameter. The dendritic processes were also shown to have a highly ordered pattern of immune complex attachment on their surface, suggestive of a periodic arrangement of receptor sites.
Chronic progressive polyarthritis and other symptoms of collagen vascular disease induced by graft-vs-host reaction.
Authors: Pals S T , Radaszkiewicz T , Roozendaal L , Gleichmann E
Published: December 2022

The induction of a GVHR in (BALB/c X A)F1 mice by i.v. injection of 80 to 120 X 10(6) BALB/c spleen cells leads to the development of chronic progressive polyarthritis, which shares several of the articular and extra-articular manifestations of human rheumatoid arthritis. The development of these lesions was found to be mediated by donor T cells and to require the presence of histoincompatibility between donor and host. The arthritis, which was mainly confined to the interphalangeal joints of the forefeet and hindfeet, was histologically characterized by periarticular and synovial lymphoid infiltrations, as well as synovial proliferation and pannus formation. Prominent juxta-articular lesions included 1) perivascular infiltrates, 2) peritendinitis, 3) myositis, and 4) inflammatory nodules. In addition, the GVH F1 mice showed pathologic symptoms reminiscent of other collagen vascular diseases, including the following: 1) a Sjögren-like salivary gland lesion, 2) lesions resembling sclerosing cholangitis, 3) scleroderma-like skin lesion, and 4) immune-complex glomerulonephritis. In most of the GVH F1 mice, these pathologic changes were accompanied by lymphoid stimulation. The spectrum of symptoms induced has many similarities to that found in mixed connective tissue disease.