bluuebunny/crossref_metadata_2025 huggingface dataset:
Decay-accelerating factor (DAF) shares a common carbohydrate determinant with the variant surface glycoprotein (VSG) of the African Trypanosoma brucei.
Authors: Davitz M A , Gurnett A M , Low M G , Turner M J , Nussenzweig V
Published: December 2022

Decay-accelerating factor (DAF) is an integral membrane protein that inhibits amplification of the complement cascade on the cell surface. We and other investigators have shown that DAF is part of a newly characterized family of proteins that are anchored to the cell membrane by phosphatidylinositol (PI). The group includes the variant surface glycoprotein (VSG) of African trypanosomes, the p63 protein of Leishmania, acetylcholinesterase (AChE), alkaline phosphatase, Thy-1, 5'-nucleotidase, and RT6.2--an alloantigen from rat T cells. The structure of the membrane anchor has been best characterized for VSG, but chemical studies of the membrane anchors of AChE and Thy-1 suggest that similar glycolipid moieties anchor these proteins to the cell surface. In the VSG, the membrane anchor consists of an ethanolamine linked covalently to an oligosaccharide and glucosamine; the entire complex is anchored to the cell membrane by PI. Immunologically, this glycolipid defines an epitope, the cross-reacting determinant (CRD), that is only revealed after removal of the diacyl glycerol anchor by a phospholipase C. By Western blotting, we show here that DAF-S (DAF released from the membrane by PI-specific phospholipase C [PIPLC]) also contains CRD. Using a newly developed immunoradiometric assay (IRMA) in which the solid-phase capturing antibody is a monoclonal antibody to DAF and the second antibody is anti-CRD, we have been able to quantitate DAF-S. By IRMA, we show that the reaction between anti-CRD and DAF-S is specific, since the binding is competitively inhibited only by the soluble form of the VSG. These observations further support the concept that the glycolipid anchors of this new family of proteins have similar structures. DAF is also found as a soluble protein in various tissue fluids as well as in Hela cell supernatants. No evidence for the presence of the CRD epitope was found on these proteins, suggesting that these forms of DAF are not released from the surface of cells by endogenous phospholipases.
Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis.
Authors: Zhang Y X , Stewart S , Joseph T , Taylor H R , Caldwell H D
Published: December 2022

A panel of monoclonal antibodies (MAb) was generated against Chlamydia trachomatis serovar B, an etiologic agent of blinding trachoma. The specificities of MAb were determined by dot blot assay by using viable elementary bodies of 13 C. trachomatis serovars and two C. psittaci strains. The dot blot assay was used to identify those antigens that were unique and immunoaccessible on the chlamydial surface. MAb were identified that recognized bi-specific (serovars B and Ba) or subspecies-specific (various B complex serovars) surface-exposed antigenic determinants that were either resistant or sensitive to heat denaturation (56 degrees C, 30 min). All of the MAb recognized the major outer membrane protein as determined by either immunoblotting or radioimmunoprecipitation. MAb specific for immunoaccessible major outer membrane protein epitopes protected mice from toxic death after i.v. injection of B serovar elementary bodies and neutralized the infectivity of the organism for monkey eyes. In contrast, MAb reactive against non-immunoaccessible subspecies- or species-specific major outer membrane protein epitopes or against an immunoaccessible genus-specific epitope located on chlamydial lipopolysaccharide did not protect mice from toxic death or neutralize infectivity of the parasite for monkey eyes. These data suggest that those major outer membrane protein antigenic determinants that are serovar or serogroup specific and are accessible to antibody on the chlamydial cell surface may be useful as a recombinant subunit vaccine for trachoma.
Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes.
Authors: Feinman R , Henriksen-DeStefano D , Tsujimoto M , Vilcek J
Published: December 2022

The mechanism of human peripheral blood monocyte-mediated cytotoxicity for tumor cells was investigated, using the A673 human rhabdomyosarcoma and HT-29 human colon adenocarcinoma lines as target cells. A673 cells were shown to be susceptible to the cytotoxic action of purified recombinant human tumor necrosis factor (TNF). A673 cells were also highly sensitive to the cytotoxic action of peripheral blood monocytes. Clones of A673 cells sensitive and resistant to TNF were isolated and characterized for their sensitivity to monocyte killing. A good correlation was found between the sensitivity of these clones to the cytotoxicity of TNF and their susceptibility to killing by monocytes. A TNF-specific neutralizing monoclonal antibody (MAb) reduced monocyte killing of parental A673 cells and of a TNF-sensitive clone of A673 cells. Inhibition of monocyte killing by this MAb was particularly pronounced at a low effector to target cell ratio. HT-29 cells were relatively resistant to the cytotoxic action of recombinant TNF and to monocyte killing. Treatment of HT-29 cells with recombinant human IFN-gamma increased their susceptibility to both TNF cytotoxicity and monocyte killing. In addition, MAb to TNF inhibited monocyte killing in HT-29 cells sensitized by incubation with IFN-gamma. Our data show that TNF is an important mediator of the cytotoxicity of human monocytes for tumor cells and that IFN-gamma can increase monocyte cytotoxicity by sensitizing target cells to the lytic action of TNF.
The cytoplasmic expression of CD3 antigens in normal and malignant cells of the T lymphoid lineage.
Authors: Campana D , Thompson J S , Amlot P , Brown S , Janossy G
Published: December 2022

Anti-CD3 (T3) Ab reacting with different proportions of thymocytes (anti-CD3a: UCHT1, anti-CD3b: T10B9, and anti-CD3c: OKT3) were tested for cytoplasmic (cCD3) and membrane (mCD3) expression in the bone marrow, thymus, and blood in man and selected primates. The expression of cCD3a and cCD3c in the perinuclear and Golgi area of large, BrdU-incorporating, strongly TdT+ thymic blasts probably represents one of the earliest signs of T cell commitment, because these blast cells are CD1-, CD4-, CD8-, and mCD3-. The cCD3+, TdT+ cells are normally restricted to the thymus and are absent among the TdT+ cells of bone marrow. The anti-CD3b Ab used, T10B9, co-caps and co-modulates with the other anti-CD3 Ab and is a T cell-specific reagent at a membrane level but does not bind to perinuclear cCD3. Instead, this reagent cross-reacts with a filamentous cytoplasmic network in non-T cells in man and in primates S. oedipus and M. rhesus despite their T cell negativity for mCD3. The characteristics of all T-ALL cases studied: cCD3+, CD7+ along with nuclear TdT+ suggest lineage fidelity to early thymic blasts. As a marked contrast, cCD3 is absent in common ALL and in AML, including cases that concomitantly express CD7 and myeloid antigens. Thus, the cCD3, TdT combination provides a very sensitive assay for residual T-ALL blasts outside the normal thymus.
Analysis of two distinct B cell activation pathways mediated by a monoclonal T helper cell. I. MHC-restricted activation of B cells by an IL 2-dependent pathway.
Authors: Asano Y , Nakayama T , Kubo M , Nakanishi K , Hodes R J , Tada T
Published: December 2022

The present study was carried out to determine whether the MHC-restricted and MHC-unrestricted B cell activation pathways mediated by a single cloned Th cell are separable, and whether these two pathways are mediated by distinct mechanisms. It was demonstrated that the two B cell activating functions of a single cloned Th cell could be separated by their sensitivity to irradiation. It was shown that MHC-restricted B cell activation is mediated by a radiosensitive Th cell function, whereas MHC-unrestricted B cell activation is mediated by a radioresistant function of the same Th cell. In addition, it was shown that recombinant IL 2 can restore or replace the radiosensitive component of MHC-restricted cognate helper function.
Activation of human B cell proliferation through surface Bp35 (CD20) polypeptides or immunoglobulin receptors.
Authors: Clark E A , Shu G
Published: December 2022

Human B cells can be activated with monoclonal antibodies (mAb) to surface IgM receptors or mAb to a 35-kilodalton B cell differentiation antigen, Bp35 (CD20). We compared anti-Ig-induced B cell activation with B cell triggering by anti-Bp35. Both anti-Ig- and anti-Bp35-dependent proliferation were augmented by the same co-stimulants, including a partially purified BCGF, recombinant IL 1, TPA, or each other. When anti-Bp35 and anti-Ig were used together to induce proliferation of tonsillar B cells, the strongest response was observed when anti-Bp35 was added 12 to 24 hr before anti-Ig. Anti-Bp35 also was found to act most effectively when added before the BCGF. Blood and tonsillar B cells differed in their proliferative response to anti-Ig or anti-Bp35: unlike dense tonsillar B cells, which consistently proliferated in response to either stimulus, blood B cells from many donors proliferated in response to anti-Ig but not to anti-Bp35 even in the presence of other co-stimuli. Dense tonsillar B cells that proliferate in response to anti-Bp35 appeared to be at a more activated stage than unresponsive blood B cells because they expressed higher levels of HLA class II molecules than blood B cells. Pretreatment of blood B cells with anti-Bp35 converted them to an HLA-DR(bri) phenotype and made them more responsive to anti-Ig-induced proliferation. These results suggest that B cells at different stages of differentiation differ in their response to anti-Bp35 and anti-Ig. The Bp35 surface polypeptide may play an early role in the activation of B cells prior to antigen or other signals.
Distribution of histopathology and Ia positive cells in actively induced and passively transferred experimental autoimmune orchitis.
Authors: Tung K S , Yule T D , Mahi-Brown C A , Listrom M B
Published: December 2022

Histopathology in testes from mice with actively induced experimental orchitis (EAO) (active EAO) and those from recipients of testis-sensitized lymphocytes (passive EAO) had different distributions. In passive EAO, maximum orchitis existed in the straight tubules, rete testis, and ductus efferentes, obstruction of which led to extreme dilatation of seminiferous tubules. Unusual intralymphatic granulomata also resulted in dilated testicular lymphatics. In active EAO, maximum orchitis affected seminiferous tubules under the testicular capsule, away from the rete testes. Vasitis was common and occurred in both active and passive EAO. In normal testes, IA+ F4/80+ cells were sparse but formed a cuff around the straight tubules. After immunization with testis in adjuvant or with adjuvant alone, the number, size, and staining intensity of IA+ cells increased dramatically beginning on day 5, 7 days before disease onset. Simultaneously, epithelial cells confined to the ductus efferentes became Ia+. Although recipients of sensitized lymphocytes also developed epithelial Ia in the ductus efferentes, they did not show changes in testicular interstitial Ia+ cells. Our findings indicate that testicular autoantigens are not completely sequestered, but are accessible to and can react with passively transferred immune lymphocytes in well-defined regions of the germ cell compartment. These regions coincided to a large extent with maximum expression of periductal or epithelial Ia. Changes in Ia+ cells in the testis, which are inducible by adjuvants and precede orchitis, may account in part for the different distribution of histopathology of active EAO.
Immune interferon enhances functional properties of human granulocytes: role of Fc receptors and effect of lymphotoxin, tumor necrosis factor, and granulocyte-macrophage colony-stimulating factor.
Authors: Perussia B , Kobayashi M , Rossi M E , Anegon I , Trinchieri G
Published: December 2022

We report here a comparative study of the effects of several cytokines known to affect myeloid cell differentiation on functional properties of human mature granulocytes. We show that recombinant interferon-gamma (rIFN-gamma), recombinant granulocyte/macrophage-colony stimulating factor (rGM-CSF), recombinant tumor necrosis factor (rTNF), and lymphotoxin (LT) purified to homogeneity are potent stimulators of polymorphonuclear cells (PMN) activity. All cytokines enhance antibody-dependent cell-mediated cytotoxicity (Ab-CMC) mediated by human PMN; however, rGM-CSF, rTNF, and LT have an immediate and short-lived effect on the PMN, whereas the activation by rIFN-gamma requires several hours of induction but can be observed up to 24 to 48 hr of culture. Only the effect of rIFN-gamma is in part dependent on induction of a high-affinity FcR for monomeric IgG on PMN, as suggested by two-color sorting analysis, and on mechanisms that result in prolonged survival of PMN in a functionally active state to mediate oxidative burst, phagocytosis, and bactericidal activity. Greater enhancement of Ab-CMC is obtained by using rIFN-gamma in combination with the other cytokines. Our data indicate that cytokines previously defined on the basis of their cytotoxic effects mediate a wide spectrum of activities on mature myeloid cells and provide evidence for their possible role in vivo, alone or in combination with rIFN-gamma, in modulating functional activities of cells responsible for non-adaptive systems of defense.
Polyclonal B cell activation by a B cell differentiation factor, B151-TRF2. III. B151-TRF2 as a B cell differentiation factor closely associated with autoimmune disease.
Authors: Dobashi K , Ono S , Murakami S , Takahama Y , Katoh Y , Hamaoka T
Published: December 2022

We demonstrated previously that B151K12 T cell hybridoma produces two distinct B cell differentiation factors, B151-TRF1 and B151-TRF2, capable of inducing differentiation of antigen-activated and unstimulated B cells into antibody-forming cells, respectively. In the present study we investigated the pathophysiologic relation of these factors with factors obtained from MRL/MP-lpr/lpr(MRL/lpr) mice and (C57BL/6 X DBA/2)F1 (BDF1) mice undergoing chronic graft-vs-host reaction (GVHR), representing a murine model of systemic lupus erythematosus with polyclonal B cell activation associated with the T cell hyperfunction. The functional and biochemical analyses revealed that B151-TRF2-like, but not B151-TRF1-like, activity was found in culture fluid supernatant (CFS) of lymphoid cells from MRL/lpr mice with lymphoproliferative syndrome. On the other hand, both B151-TRF1- and B151-TRF2-like activities were detected in CFS prepared from spleen cells of BDF1 mice undergoing chronic GVHR by the inoculation of parental DBA/2 spleen cells. Interestingly, spleen cells of BDF1 mice transferred with DBA/2 thymocytes preferentially elaborated B151-TRF1-like factor. Because BDF1 mice transferred with DBA/2 spleen cells but not with DBA/2 thymocytes developed a SLE-like syndrome exemplified by the appearance of Coombs' antibody and proteinuria, it seemed likely that production of B151-TRF2-like factor was closely associated with the onset of autoimmune disease. In fact, B151-CFS containing B151-TRF2 but not B151-TRF1 activity could induce a striking autoantibody production both in vivo and in vitro as detected by PFC responses of normal mice to bromelain-treated mouse red blood cells (BrMRBC). Moreover, it was demonstrated that in vitro anti-BrMRBC PFC responses induced by semipurified B151-TRF2 was markedly inhibited by addition of relevant anti-Ia antibody to the culture. Thus, the present study demonstrates that B151-TRF2 represents one of the B cell differentiation factors responsible for polyclonal B cell activation leading to autoantibody production.
Augmentation of normal and malignant B cell proliferation by monoclonal antibody to the B cell-specific antigen BP50 (CDW40).
Authors: Ledbetter J A , Shu G , Gallagher M , Clark E A
Published: December 2022

We recently described a 50,000 dalton polypeptide Bp50 (CDw40) that is expressed on human B cells and plays a role in regulating B cell proliferation. Here we additionally characterize the functional signal given by antibody binding to Bp50 on both normal and malignant B cells. A monoclonal anti-Bp50 antibody could augment the proliferation of B cells activated by anti-IgM, anti-CD20, or 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulation, but was not co-stimulatory with B cell growth factor (BCGF), interleukin 1, or interleukin 2. The signal did not depend on the Fc portion of the antibody, because F(ab')2 fragments of anti-Bp50 were still functionally active. Both anti-Bp50 and a low m.w. BCGF preparation were similar in that both were co-stimulatory with the same agents and both anti-Bp50 and BCGF affected activated B cells but not resting B cells. However, a panel of B cell malignancies differed in their responsiveness to anti-Bp50 vs BCGF: some tumors proliferated in response to anti-Bp50 but not BCGF, whereas other tumors had the opposite pattern. Bp50 was found to have several properties in common with HLA class II molecules: both Bp50 and class II were expressed at lower levels on blood B cells than on tonsillar B cells; the expression of both Bp50 and class II was increased after activation of blood B cells with TPA or anti-IgM; and the expression of both Bp50 and class II was increased after activation of non T, non-B acute leukemias with BCGF. Thus class II and Bp50 expression may be under common regulatory control. The fact that BCGF modulated the expression of Bp50 on leukemic cells suggests that BCGF and Bp50-mediated signals may be coordinately regulated.
Identification of a novel T cell surface disulfide-bonded dimer distinct from the alpha/beta antigen receptor.
Authors: Nagasawa R , Gross J , Kanagawa O , Townsend K , Lanier L L , Chiller J , Allison J P
Published: December 2022

Hybridomas were prepared from the spleen of a BALB/c mouse immunized with EL-4 T lymphoma cells. One, designated A1, was found to secrete a monoclonal antibody that reacted with two T lymphoma cells of C57BL origin, EL-4 and C6VLB, but not with normal C57BL/6 splenocytes or thymocytes, C57BL/6 T cell clones, or other T or B lymphomas by complement-mediated cytotoxicity or indirect immunofluorescent staining. Monoclonal antibody (MAb) A1 precipitated a protein that migrated at 85 kD under nonreducing and 43 kD under reducing conditions. The fact that the antigen defined by MAb A1 was a disulfide-linked dimer, together with the essentially clone-specific distribution of the reactive epitope, raised the possibility that the antibody defined an epitope of the antigen receptor. However, several additional observations revealed that the antibody defined a distinct and novel T cell surface structure. MAb 124-40, previously shown to react with the antigen receptor of C6VLB cells, reacted with variants of C6VLB that failed to express the A1 epitope. Sequential immunoprecipitation indicated that MAb A1 and MAb 124-40 reacted with distinct molecular species on C6VLB cells. Endoglycosidase digestion showed that the structure reactive with MAb A1 was not derived from that reactive with MAb 124-40 by addition of N-linked oligosaccharide residues. Two-dimensional gel electrophoretic analysis of precipitates obtained from radioiodinated C6VLB cells with MAb 124-40 resolved the alpha and beta subunits of the antigen receptor. Similar analysis of precipitates obtained with MAb A1 revealed only a single basic chain under reducing conditions, although anomalous mobility suggestive of a second, more acidic chain was observed under nonreducing conditions. Two-dimensional maps of tyrosine-containing chymotryptic peptides of the proteins isolated with MAb A1 and MAb 124-40 were completely different, suggesting that the molecules shared no peptides and were distinct in primary structure. Finally, cross-linking studies performed with a cleavable reagent indicated that the A1 molecule, unlike the antigen receptor defined with MAb 124-40, was not associated with additional, T3-like structures on the surface of C6VLB cells. Although the MAb A1 was unreactive with normal cells in cytotoxicity or staining assays, a molecule of the appropriate size was immunoprecipitated in small amounts from lysates of radioiodinated normal spleen and thymus cells. These data indicate that MAb A1 defines a novel disulfide-linked T cell surface molecule distinct from the antigen receptor.
Multimeric C9 within C5b-9 is required for inner membrane damage to Escherichia coli J5 during complement killing.
Authors: Bloch E F , Schmetz M A , Foulds J , Hammer C H , Frank M M , Joiner K A
Published: December 2022

We have shown recently that an average of three or more C9 molecules must bind to C5b-8 on Escherichia coli strain J5 to cause direct complement killing in the absence of serum lysozyme. We initially confirmed and extended this observation by showing that deposition of a large number of C5b-9 complexes bearing 1C9 per C5b-8 was not bactericidal for J5. To identify the target site for bactericidal C5b-9 deposition, we measured release of periplasmic and cytoplasmic markers of different size from J5 as the C9:C5b-8 ratio was changed, because the diameter of the C5b-9 channel is known to increase as the C9:C5b-8 ratio increases. To facilitate measurement of release of the periplasmic marker beta-lactamase (BLA), J5 was transformed for high level constitutive TEM-1 BLA production (J5-Amp). Multimeric C9 within C5b-9 (C9:C5b-8 greater than 3) was required to release BLA (m.w. 28,900) from J5-Amp regardless of whether cells bore 310, 560, or 890 C5b-9/organism. Curves of both BLA release and killing vs C9:C5b-8 ratio were sigmoidal and nearly superimposable. Release of the small cytoplasmic marker 86Rb, a potassium analog, also required a minimum C9:C5b-8 ratio of 3:1; specific 86Rb release did not occur in the absence of killing. Release of the large cytoplasmic marker beta-galactosidase (m.w. 505,000) did not occur even at the highest achievable C9:C5b-8 ratio of 11:1, despite greater than 99.9% killing, indicating that there was no dissolution of the peptidoglycan layer due to incomplete removal of serum lysozyme. Complement-mediated killing of J5 requires sufficient damage to the outer membrane or formation of a sufficiently large C5b-9 channel to release the large periplasmic marker BLA. The requirement of multimeric C9 for 86Rb release suggests that at low C9:C5b-8 ratios, either C5b-9 does not have access to the cytoplasmic space or that the J5 K+ transport systems are able to compensate for putative C5b-9 channels.
Constitutive and IL 1-regulated murine complement gene expression is strain and tissue specific.
Authors: Falus A , Beuscher H U , Auerbach H S , Colten H R
Published: December 2022

To study the molecular mechanisms accounting for strain- and tissue-specific variations in the production of complement proteins, complementary DNA probes were used to assess qualitative and quantitative differences in specific mRNA content of complement proteins C2, factor B, and C3 in extracts of tissues (liver, lung, spleen, kidney, and peritoneal macrophages) isolated from various mouse strains. Northern blot analysis of total hepatic RNA revealed differences in C2, factor B, and C3 mRNA levels in strains that share B10 background but differ in the H-2 region (e.g., H-2k, H-2u, H-2d, H-2f). In each instance, hepatic mRNA specific for the individual gene product corresponded in amount to the serum levels. By contrast, specific mRNA content of C2 and factor B in macrophages differed significantly from those observed in liver for each strain. Modulation of C2, factor B, and C3 expression was studied after in vivo administration of recombinant IL 1 or endotoxin to H-2k (B10.AKM) or H-2u (B10.PL) strain mice. As assessed by Northern blot analysis, neither endotoxin nor IL 1 affected liver C2-specific mRNA but increased specific C2 mRNA levels in kidney and lung. For both strains, IL 1 increased specific factor B mRNA in all tissues examined except for the H-2u strain liver factor B mRNA content, which was not affected by IL 1, whereas that of H-2k mice was increased. The lack of factor B modulation by IL 1 in the H-2u lines was specific to that gene and not a reflection of a generalized IL 1 unresponsiveness. Differences in tissue and strain specific constitutive and IL 1-regulated expression of the C3 gene were also observed in the H-2u and H-2k strains.
Accumulation and chemotaxis of natural killer/large granular lymphocytes at sites of virus replication.
Authors: Natuk R J , Welsh R M
Published: December 2022

A model for monitoring the accumulation of natural killer cell/large granular lymphocytes (NK/LGL) at a site of virus replication was studied by using mice infected i.p. with either lymphocytic choriomeningitis virus (LCMV), murine cytomegalovirus (MCMV), mouse hepatitis virus (MHV), Pichinde virus, or vaccinia virus. An i.p. but not i.v. infection resulted in a localized increase in NK/LGL cell number (a fourfold to greater than 20-fold increase) and augmentation (a 10- to 20-fold increase) of NK cell activity associated with virus-induced peritoneal exudate cell (PEC) populations. An increase in NK/LGL cell number was detected as early as 12 hr postinfection (p.i.) and peaked at 3 days p.i. with MHV. The initial LGL recruited into the peritoneal cavity at 1 to 3 days p.i. were nonadherent to plastic and were demonstrated to have an NK cell phenotype: asialo GM1+, Thy-1.2 +/-, Lyt-2.2-, and J11d-. The peak number of LGL appeared at 7 days after infection with the NK cell-resistant virus, LCMV. This LGL population had been previously demonstrated to contain cytotoxic T lymphocyte/LGL (CTL/LGL) as well as NK/LGL. During an MHV infection the number of LGL decreased between days 3 and 7 p.i., suggesting that the second wave of CTL/LGL was absent. These findings may explain the absence of a good MHV-CTL model. Virus-induced, activated NK/LGL responded to chemotactic signals by migrating in a unidirectional manner across two 5-microns pore size polycarbonate filters during 7 hr in vitro chemotaxis assays. Wash-out fluid obtained from the peritoneal cavity contained chemotactic activity for NK/LGL as well as for other cell types. We conclude that production and/or release of chemotactic factors at sites of virus replication are at least partially responsible for the accumulation of NK/LGL at these sites.
Role of NK cells in protection of mice against herpes simplex virus-1 infection.
Authors: Rager-Zisman B , Quan P C , Rosner M , Moller J R , Bloom B R
Published: December 2022

Natural killer (NK) cells have been implicated in the recognition and killing of a variety of virus infected target cells in vitro, yet their role in vivo remains uncertain. In these experiments, the role of NK cells in the regulation of resistance to herpes simplex virus-1 (HSV-1) was studied. Adult C57BL/6 mice are resistant to HSV-1 (HFEM strain), but are rendered highly susceptible by treatment with cyclophosphamide 24 hr prior to infection. In this model, passive transfer of 10(8) normal spleen cells or 10(7) poly I:C-treated spleen cells provided protection for 72% of the recipients. Spleen cells from NK cell-deficient beige mice similarly treated failed to engender passive protection. The phenotype of the cells responsible for transferring protection was NK1.1+, and asialo GM1+. Transfer of NK cells resulted in marked reduction of HSV titers in the livers and brains of recipients. These experiments provide direct evidence for a role for NK cells in protection against development of fatal HSV infection in mice.
Lipopolysaccharide-mediated macrophage activation: the role of calcium in the generation of tumoricidal activity.
Authors: Drysdale B E , Yapundich R A , Shin M L , Shin H S
Published: December 2022

As we have reported, calcium ionophore A23187 activates macrophages for tumor cell killing, and the activated macrophages produced a soluble cytotoxic factor (M phi-CF) that is similar, if not identical, to tumor necrosis factor. Based on these observations, we have investigated whether calcium is involved in the activation mediated by another potent macrophage activator, namely lipopolysaccharide (LPS). We first showed that A23187 caused uptake of extracellular calcium-45 by macrophage monolayers, whereas LPS did not. Because in this system rapid changes would not have been detected, several other approaches also have been used. We have examined the effect of depleting extracellular calcium by using medium containing no added calcium, supplemented with 1 mM EGTA. In no case did depletion result in decreased M phi-CF production by LPS-treated macrophages. Measurements using the fluorescent intracellular calcium indicator Quin 2 have also been performed. The calcium ionophore ionomycin caused a rapid change in the intracellular Quin 2 signal. LPS, even at a concentration in vast excess of that required to activate the macrophages, caused no change in the signal during a 2-hr period. If the macrophages were loaded with high doses of Quin 2 or another intracellular chelator, TMB-8, M phi-CF production decreased and cytotoxic activity was impaired. These data indicate that one or more of the processes involved in M phi-CF production does require calcium, but that activation mediated by LPS occurs without the influx of extracellular calcium or redistribution of intracellular calcium.
The B cell is the initiating antigen-presenting cell in peripheral lymph nodes.
Authors: Janeway C A , Ron J , Katz M E
Published: December 2022

We have examined the role of B cells in antigen presentation in lymph nodes in several ways. We found that mice depleted of B lymphocytes via chronic injection of anti-mu-chain antibody do not mount peripheral lymph node T cell proliferative responses to normally immunogenic doses of antigen. Depletion of B cells by passage of immune lymph node cells over anti-immunoglobulin columns early after immunization depletes antigen-presenting function from draining lymph nodes, and this function can be restored by using B cells or splenic adherent cells to allow the remaining T cells to proliferate. Lymph node B cells present antigen very effectively to lines of antigen-specific T cells. However, unfractionated lymph node cells from anti-mu-treated mice present very poorly, if at all, whereas unfractionated spleen cells from the same mice do present antigen. This is in keeping with our previous finding that helper T cell function in the spleen is normal in B cell-deprived mice. Finally, when mice homozygous for the lymphoproliferative gene lpr are treated chronically with anti-mu-chain antibody, lymphadenopathy is greatly retarded, suggesting a role for B cells in the massive proliferation of T cells in this syndrome. From this analysis, it would appear that the initiating antigen-presenting cell in the lymph node is a B lymphocyte, and that B lymphocytes in lymph nodes may be distinct from those in the spleen. It is of interest that these results also suggest that the lymph node lacks an antigen-presenting cell that is found in the spleen, perhaps the dendritic cell.
Antipeptide antibodies define the NH2-terminal structure of the pan-specific hemopoietin interleukin 3.
Authors: Ziltener H J , Clark-Lewis I , Fazekas de St Groth B , Hood L E , Kent S B , Schrader J W
Published: December 2022

Antipeptide antibodies were raised in rabbits against a synthetic peptide including the six NH2-terminal amino acids of the pan-specific hemopoietin interleukin 3 (IL 3). Affinity-purified antibody preparations specific for epitopes determined by residues 1 to 6 were immobilized and used as affinity columns. Up to 98% of IL 3 bioactivity in T cell-conditioned medium was depleted by these columns, as was 71 and 74%, respectively, of IL 3 aberrantly produced by the myeloid leukemias WEHI-274.14 and WEHI-3B. IL 3 produced in vivo in WEHI-3B tumor-bearing mice also bound to the anti-1-6 antibody column, up to 70% of the bioactivity being depleted from ascites fluid and up to 84% from the serum. These results suggest that all IL 3 secreted by T cells and the majority of the IL 3 molecules secreted by myeloid leukemias express epitopes determined by residues 1-6 and cannot have the NH2-terminal amino acid sequence initially reported for IL 3. These six NH2-terminal amino acids share similarities with the NH2-terminal amino acids of several other lymphokines, suggesting an important function for this hexapeptide.
Expression in Escherichia coli of fully active recombinant human IL 1 beta: comparison with native human IL 1 beta.
Authors: Tocci M J , Hutchinson N I , Cameron P M , Kirk K E , Norman D J , Chin J , Rupp E A , Limjuco G A , Bonilla-Argudo V M , Schmidt J A
Published: December 2022

Although complementary DNA (cDNA) encoding human interleukin 1 beta (IL 1 beta) have been cloned in several laboratories, there are as yet no data demonstrating that recombinant IL 1 beta (rIL 1 beta) molecules expressed from such cDNA are faithful, fully active replicas of the native protein secreted by human monocytes. To this purpose, cDNA sequences corresponding to the exact NH2-terminus and amino acid sequence of mature, monocyte-derived human IL 1 beta were placed under control of the inducible trp-lac (TAC) fusion promoter and were expressed in E. coli strain JM105. rIL 1 beta was purified to homogeneity by high pressure liquid chromatography (HPLC). Yields of 10 to 20 mg of rIL 1 beta/liter/10(11) cells were obtained. Purified rIL 1 beta was then compared in a number of biochemical and biologic assays to purified native IL 1 beta. Native and rIL 1 beta co-migrated on SDS-polyacrylamide gels as 17.5 kd polypeptides and reacted with specific polyclonal antisera raised to three synthetic peptides of human IL 1 beta in immunoblot experiments. Amino acid sequence analysis of rIL 1 beta showed that the native amino terminus, an ALA residue, was faithfully maintained. Purified native and rIL 1 beta co-chromatographed on reverse-phase HPLC. Specific biologic activities of rIL 1 beta were indistinguishable from those of the native protein in murine thymocyte and human dermal fibroblast proliferation assays, with half-maximal stimulation occurring at concentrations of 25 pM in the murine thymocyte assay and 2 pM in the human dermal fibroblast assay. Similarly, native and rIL 1 beta competed equally well for high affinity IL 1 receptor binding sites, each exhibiting a Ki of 20 pM on MRC-5 human embryonic lung fibroblasts. These observations indicate that E. coli efficiently expresses large quantities of rIL 1 beta, which emulates exactly the properties of the native protein.
Probing functional sites on complement protein B with monoclonal antibodies. Evidence for C3b-binding sites on Ba.
Authors: Ueda A , Kearney J F , Roux K H , Volanakis J E
Published: December 2022

We used four mouse monoclonal antibodies (Mab) as probes of functional sites of human complement protein B. Two Mab, HA4-1B (gamma 2a kappa) and HA4-15 (gamma 2a kappa), reacted with the same or adjacent epitopes on the Bb fragment of B, while the other two, HA4-1A (gamma 1 kappa) and FD3-20 (gamma 1 kappa), reacted with distinct epitopes on Ba. All reactive epitopes were expressed on native B and only one, recognized by the anti-Ba Mab HA4-1A was more reactive on isolated Ba than on B. These binding specificities were determined by direct binding radioassays and confirmed by inhibition studies. Immunoelectron microscopy of B and Bb in complex with anti-Ba and anti-Bb revealed that the recognized epitopes are on opposite sides of the molecule and are on discrete domains. All four Mab inhibited the hemolytic activity of B, although with different efficiencies and through different mechanisms. The main effect of the two anti-Bb Mab was an increased rate of loss of hemolytic sites from preformed EC3bBb C3 convertase presumably through accelerated dissociation of Bb. On the other hand, the main effect of the two anti-Ba Mab was inhibition of binding of B to C3b. HA4-1A was more efficient, inhibiting by 50% the binding of [125I]B to EC3b at 10 micrograms/ml as IgG and at 13 micrograms/ml as Fab. The data suggest that a binding site for C3b on intact B is located on the Ba portion of the molecule.
Modulation of FcR function by complement: subcomponent C1q enhances the phagocytosis of IgG-opsonized targets by human monocytes and culture-derived macrophages.
Authors: Bobak D A , Gaither T A , Frank M M , Tenner A J
Published: December 2022

We have investigated the interaction of C1q, a subunit of the first component of complement, with human monocytes and culture-derived macrophages. Adherence of these mononuclear phagocytes to surfaces coated with C1q induced a marked enhancement of the phagocytosis of sheep erythrocytes opsonized with IgG anti-Forssman antibody (EA-IgG). This C1q-mediated enhancement of phagocytosis was dose dependent, and was specifically blocked by pretreatment of the C1q-coated surfaces with F(ab')2 anti-C1q. The augmentation of FcR-mediated phagocytosis by C1q was determined to be a result of the interaction between the C1q and the phagocytic effector cell, and was not due to interaction between the surface-bound C1q and the EA-IgG. Neither resting nor N-formyl-methionyl-leucyl-phenylalanine-stimulated polymorphonuclear leukocytes were induced by C1q to increase FcR-mediated phagocytosis. Experiments conducted with purified fragments of C1q suggest that the C1q phagocytosis enhancement signal resides in the collagen-like tail domain of the molecule. This region is the same portion of the molecule previously shown to interact with the cell surface C1q receptor. Native type I collagen was unable to enhance FcR-mediated phagocytosis by mononuclear phagocytes. It has been demonstrated that C1q can be localized to areas of inflammation, and additionally C1q can be secreted by macrophages in culture. In view of these findings and the results of our present study, we hypothesize that C1q could provide local, direct, and non-opsonic enhancement of phagocytosis by mononuclear phagocytes in areas of infection and inflammation.
Arsonate-specific murine T cell clones. IV. Properties of I-E- and I-A-restricted clones.
Authors: Spragg J H , Goodman J W
Published: December 2022

The T cell antigen L-tyrosine-p-azobenzenearsonate is unique in being a simple determinant that can be presented in the context of both I-A and I-E. I-E-restricted T cell clones derived from B10.A(5R) mice were found to fall into three groups: Type I clones recognized antigen only in the context of syngeneic apcs, Type II clones recognized antigen with the same highly specific major histocompatibility complex restriction but in addition proliferated in response to allogeneic stimuli; Type III clones were "degenerate" in their major histocompatibility complex-restricted recognition of antigen and proliferated when antigen-presenting cells bearing Eb beta Ek alpha (syngeneic), Ek beta Ek alpha, or Ed beta Ed alpha were used. These observations allow some conclusions to be drawn about sites on the I-E molecule that may be functionally significant in the presentation of this antigen. By using the B cell hybridoma LK35.2 as target cells, some of these T cell clones act as cytotoxic cells in the Class II-restricted manner predicted from the results of proliferative assays. Class II-restricted cytotoxicity can therefore be controlled by both I-A and I-E mouse Ir gene loci.
Recognition of human polymorphonuclear leukocyte receptors for leukotriene B4 by rabbit anti-idiotypic antibodies to a mouse monoclonal antileukotriene B4.
Authors: Gifford L A , Chernov-Rogan T , Harvey J P , Koo C H , Goldman D W , Goetzl E J
Published: December 2022

Rabbit anti-idiotypic IgG antibodies to the combining site of a mouse monoclonal IgG2b antibody to leukotriene B4 (LTB4) cross-reacted with human polymorphonuclear (PMN) leukocyte receptors for LTB4. Anti-idiotypic IgG and Fab both inhibited the binding of [3H]LTB4, but not [3H]N-formylmethionyl-leucylphenylalanine (fMLP), to PMN leukocytes with similar concentration-effect relationships, whereas neither nonimmune rabbit IgG nor Fab had any inhibitory activity. At a concentration of anti-idiotypic IgG that inhibited by 50% the binding of [3H] LTB4 to PMN leukocytes, the antibodies preferentially recognized high affinity receptors. Anti-idiotypic IgG and Fab inhibited PMN leukocyte chemotactic responses to LTB4, but not fMLP, with concentration-effect relationships resembling those characteristic of the inhibition of binding of [3H] LTB4, without altering the LTB4-induced release of beta-glucuronidase. Chemotaxis and increases in the cytoplasmic concentration of calcium equal in magnitude to those elicited by optimal concentrations of LTB4 were attained at respective concentrations of anti-idiotypic IgG equal to and 1/25 the level required for inhibition of binding of [3H]LTB4 by approximately 50%. Thus, the anti-idiotypic antibodies bound to PMN leukocyte receptors for LTB4 with a specificity, preference for high affinity sites, and capacity to alter PMN leukocyte functions that were similar to LTB4.
Biochemical characterization of a mast cell plasma membrane antigen shared by mouse serosal, culture-derived, and virally transformed mast cells.
Authors: Katz H R , Austen K F
Published: December 2022

The expression of the antigenic determinant identified by the B54.2 rat monoclonal antibody on four populations of mouse mast cells has been quantified, and the epitope-bearing surface antigen and its biosynthesis have been characterized. As assessed by indirect immunofluorescence staining and flow cytometric analysis, B54.2 antibody bound to serosal mast cells (S-MC), bone marrow culture-derived mast cells (BM-MC), fetal liver culture-derived mast cells (FTL-MC), and Abelson murine leukemia virus-transformed FTL-MC (ABFTL-MC). However, the intensity of cell surface fluorescence exhibited by ABFTL-MC was approximately eightfold less per cell compared with nontransformed, culture-derived mast cells. Immunoprecipitation of B54.2 antibody-binding molecules from each population of mast cells labeled intrinsically with [35S]methionine and analysis by SDS-PAGE demonstrated that the B54.2 epitope was expressed in each case on two noncovalently associated proteins of 110,000 Mr and approximately 130,000 Mr, but that the percentage of radiolabel in the latter species was approximately threefold less in ABFTL-MC than in BM-MC. As assessed by pulse-chase analysis with [35S]methionine, the 110,000 Mr protein was a precursor of the 130,000 Mr molecule ("B54.2 antigen") synthesized by BM-MC. Labeling of BM-MC with [35S]methionine in the presence of tunicamycin followed by immunoprecipitation and SDS-PAGE of B54.2 antibody-binding material revealed a single species of 93,000 Mr, indicating that the native molecules contained N-linked carbohydrate. Endoglycosidase H treatment of the glycoproteins precipitated by B54.2 antibody from BM-MC reduced the Mr of the 110,000-Mr molecule to 93,000 Mr without an appreciable change in the 130,000-Mr species. These data indicate that the 110,000-Mr precursor form is a "high mannose" type glycoprotein and the 130,000-Mr membrane surface B54.2 antigen is a "complex" type glycoprotein, and that the epitope recognized by the B54.2 antibody on the surface of the mouse mast cell populations is located on the 93,000-Mr peptide core.
A serine proteinase inhibitor produced by an HTLV I virus-transformed human T lymphocyte line.
Authors: Ganea D , Cearlock D , Minowada J , Dray S
Published: December 2022

A previous report from our laboratory indicated that a proteinase inhibitor is produced by rabbit T lymphocytes. We now report that a human T cell line, C91/PL, produces a proteinase inhibitor which inhibits the enzymatic activity of trypsin and kallikrein. This newly identified proteinase inhibitor (LPI 1) did not inhibit the enzymatic activity of four other serine proteinases (thrombin, plasmin, chymotrypsin, or pancreatic elastase), a thiol proteinase (papain), or a carboxyl proteinase (pepsin). Active synthesis of LPI 1 by the C91/PL cell line was shown by the appearance of similar levels of inhibitory activity in sequential cell supernatants, lack of appearance of inhibitor in supernatants of cells killed by heat or sodium azide or of viable cells in the presence of cyclohexamide, and incorporation of a radiolabeled amino acid into newly synthesized inhibitor. Although both the inhibitor of rabbit origin and of human origin are proteins produced by T cells and have similar inhibitory specificity, important differences were observed: LPI 1 is sensitive to boiling and the two inhibitors migrate differently upon electrophoresis in substrate-containing polyacrylamide gel. Furthermore, LPI 1 was produced by a cell line of the T4 phenotype which had been established by in vitro viral transformation of human cord blood lymphocytes with HTLV 1 whereas the inhibitor of rabbit origin was produced by normal splenic T cells. Three other human T cell lines of the T4 phenotype, MOLT-13, KE-37, and HPB-ALL, from patients with acute lymphoblastic leukemia did not produce a proteinase inhibitor. Thus, the production of proteinase inhibitors does not appear to be a general characteristic of human T cell lines nor of the T4 subset. Proteinase inhibitors produced by T cells may have an immunoregulatory role in proteinase-mediated physiological processes.
Analysis of immune function in herpes zoster patients: demonstration and characterization of suppressor cells.
Authors: Cauda R , Grossi C E , Whitley R J , Tilden A B
Published: December 2022

We sought to identify imbalances of immune regulatory cells that might contribute to the depression of cell-mediated immunity that occurs during an episode of herpes zoster. Peripheral blood mononuclear cells (PBMC) were obtained from patients with herpes zoster during the acute (less than 7 days after disease onset) and convalescent (more than 10 days after disease onset) phases of illness and from healthy seropositive donors. The PBMC were analyzed for: lymphoproliferative responses to varicella-zoster virus (VZV) antigens, Leu-3 (helper/inducer):Leu-2 (cytotoxic/suppressor) ratios, and percentages of suppressor cells as defined by coexpression of the Leu-2 and OKM1 antigens. Significantly depressed proliferative responses of VZV antigens and Leu-3:Leu-2 ratios, and increased percentages of Leu-2+ OKM1+ suppressor cells were observed in PBMC of acute phase herpes zoster patients as compared with the PBMC of convalescent patients or healthy donors. These differences were also observed in individual patients sequentially studied during both phases of disease. Cryopreserved acute phase PBMC suppressed the proliferative response of autologous convalescent phase PBMC to VZV antigens, but not to herpes simplex virus (HSV) antigens. The acute phase PBMC suppressor cell was radiation sensitive and was identified as a Leu-2+ cell by fluorescence-activated cell sorting. Thus, depression of cell-mediated immunity during the acute phase of herpes zoster was associated with a relative increase of lymphocytes expressing a suppressor cell phenotype and the activation of a radiosensitive Leu-2+ suppressor cell with some degree of antigen specificity.