bluuebunny/crossref_metadata_2025 huggingface dataset:
Recruitment of Effector Lymphocytes by Initiator Lymphocytes
Authors: Livnat Shmuel , Cohen Irun R.
Published: December 2022

We have shown previously that initiator T lymphocytes (ITL), sensitized in vitro against fibroblast antigens, recruit effector T cells in vivo. After injection into hind footpads of syngeneic recipients, sensitized ITL migrated to the draining popliteal lymph nodes (PLN) and activated a trapping mechanism by which circulating lymphocytes were recruited in the PLN. This paper reports experiments designed to test the immunospecificity of these recruited T lymphocytes (RTL). We found that immunospecific RTL were depleted from other lymphoid organs during recruitment in the PLN. However, immunospecific ITL were not depleted from spleens during PLN recruitment. Thus ITL and RTL are functionally distinguishable.

We show that specific GVH reactive lymphocytes were also lost from spleens and distal lymph nodes during trapping of RTL in the PLN. Thus, the trapping phase of the recruitment response is immunospecific, as are the sensitization and effector phases. The trapped RTL are antigen-specific, and include the pool of GVH-reactive-lymphocytes committed to the same alloantigen. Thus, it appears that GVH-reactive cells respond to syngeneic ITL sensitized against allogeneic fibroblasts.
Demonstration of Binding Sites for IgG Fc and the Third Complement Component (C3) on Isolated Hepatocytes
Authors: Hopf Uwe , Büschenfelde Karl-Hermann Meyer zum , Dierich Manfred P.
Published: December 2022

Isolated hepatocytes from rabbits with experimental acute serum sickness showed immune complexes bound to the hepatocellular membrane with a coarse granular fluorescent pattern. Also in vitro preformed immune complexes (BSA-anti-BSA) or aggregated γ-globulin from human and rabbit could be bound to the surface of isolated hepatocytes. In contrast, immune complexes with F(ab′)2 anti-BSA were not fixed on the membranes. Hepatocytes incubated in fresh serum showed membrane-fixed C3 in a coarse granular pattern. This deposition could be abolished by heating (56°C, 30 min) the serum or by adding EDTA (0.02 M). Also, purified human or guinea pig C3 could be bound to the hepatocellular membrane but in a linear fluorescent pattern. Thus, fixation of immune complexes on hepatocytes appears to operate through binding sites for IgG Fc and, possibly, also through binding sites for C3. It is suggested that these hepatocellular-binding sites may have a physiologic clearance function. In vivo fixed IgA could be detected on the membranes of isolated hepatocytes from healthy persons. It is assumed that the membrane-fixed IgA has a carrier function for antigens from the gut.
Radioimmunoassay for Thymopoietin
Authors: Goldstein Gideon
Published: December 2022

A radioimmunoassay for thymopoietin was developed that detects thymopoietin concentrations greater than 5 ng/ml. A critical point in development of the radioimmunoassay was preparation of 125I-labeled thymopoietin by the Bolton-Hunter method, since direct iodination of tyrosyl residues impaired the immunoreactivity of thymopoietin.
Histamine Release from Cultured Human Basophils: Lack of Histamine Resynthesis After Antigenic Release
Authors: Drobis Jeffrey D. , Siraganian Reuben P.
Published: December 2022

Human peripheral basophils can be maintained in culture for up to 72 hr. These cells retain their functional integrity as judged by total histamine content and by their ability to release histamine by an IgE-mediated reaction in response to a specific antigen challenge. Cells cultured after suboptimal antigenic challenge could be activated to release histamine upon the addition of antigen. In contrast, cells cultured after supra-optimal antigenic challenge did not fully recover their ability to release histamine even after 24 hr.

With a variety of culture conditions, it was impossible to demonstrate any net synthesis of histamine in cells. Cells cultured after depletion of their stores by reaction with antigen did not show any net histamine formation. The experiments suggest that the basophil in peripheral blood is functionally an end-stage cell and can participate in the histamine release reaction only once.
Modulation of Cyclic AMP in Purified Rat Mast Cells
Authors: Sullivan Timothy J. , Parker Keith L. , Kulczycki Anthony , Parker Charles W.
Published: December 2022

Changes in rat mast cell cyclic adenosine 3′,5′ monophosphate (cAMP) concentrations during stimulation of histamine release by concanavalin A (con A) and anti-IgE were studied. Con A caused an increase in cAMP with a mean peak level at 20 sec of 232% of control (range 164% to 365%). Con A-stimulated cells demonstrated falls toward control levels after 20 sec, but generally remained above control for at least 5 min. By 10 min cAMP had returned to control values. The con A effect on cAMP occurred in the absence of phosphatidyl serine but was markedly inhibited by 5 mM α-methyl-D-mannose. Anti-IgE induced a less marked increase in cAMP (157% of control, range 110% to 540% of control) which reached a peak at 20 sec. Two monospecific goat anti-rat myeloma IgE antisera induced similar changes in cAMP whereas normal goat IgG had no effect. These peak values were followed by a rapid decrease in cAMP. Within 2 min the cAMP content of anti-IgE stimulated cells had fallen to levels well below control and remained below control levels from 45 sec to over 15 min. Histamine release in both systems began after the peak cAMP levels, during the period of rapid destruction of cAMP.
Antibody-Induced Killing in Vivo of L1210/MTX-R Cells Quantitated in Passively Immunized Mice with 131I-Iododeoxyuridine-Labeled Cells and Whole-Body Measurement of Retained Radioactivity
Authors: Carlson George A. , Terres Geronimo
Published: December 2022

The killing of the LR subline of the DBA/2J leukemia L1210/MTX by passive antibody was followed in vivo with 131I-iododeoxyuridine-labeled cells and whole-body measurement of retained radioactivity. The in vivo killing of LR cells was proportional to the in vitro 2-mercaptoethanol resistant titer, independent of the complement system, and radioresistant. Although a large percentage of the leukemic cells was killed in passively immunized mice, the protective effect of the passive antiserum was dependent on the active immune response of the host.
Immune Response in the Hamster
Authors: Portis J. L. , Coe J. E.
Published: December 2022

A hemolytic plaque assay was used to quantitate the antibody response of the Syrian hamster after immunization with hen egg albumin (HEA). Whereas HEA in complete Freund's adjuvant (HEA-CF) induced a prolonged heterogeneous (IgM, IgG1 and IgG2) antibody response, the response to soluble HEA in saline (HEA-S) differed in that: 1) both the primary and secondary responses were restricted to the IgG1 class; 2) the IgG1 primary response was cyclical with PFC peaks on days 9 and 16; 3) although an anamnestic secondary response was demonstrated, no further augmentation was noted after tertiary and quaternary boosters; 4) the booster response was transient reaching a peak after 48 hr and declining to low levels within 7 days. Adoptive transfer of lymph node cells to irradiated recipients followed by challenge with HEA-CF revealed: 1) that HEA-S-treated donor cells were primed for an IgG1 response because anamnesis was seen 7 days after challenge, yet on day 21, IgG1 PFC were 20-fold less than that of controls; 2) recipients of HEA-S treated cells showed profound suppression of both IgM and IgG2 PFC on days 14 and 21. These studies indicate that soluble antigen induced in hamsters a state of complete tolerance of IgM and IgG2 classes whereas the anamnestic response of the IgG1 class remained intact.
Influenza A Virus Interaction with Murine Lymphocytes
Authors: Woodruff Judith J. , Woodruff Jack F.
Published: December 2022

This report describes changes in surface properties of rat thoracic duct lymphocytes (TDL) induced by influenza virus A/Japan 305 (H2N2). After incubation with virus at 37°C, sialic acids were released from the membranes of TDL with concomitant reduction in their mean electrophoretic mobility. Autoradiographs of TDL which had been incubated with 125I virus at 4°C showed that the particles attached to nearly all the lymphocytes although there was variation in the amount of virus bound per cell. Attachment occurred rapidly with maximum labeling evident after incubation at 4°C for 5 min. Incubation at 37°C for 1 hr resulted in a 10 to 18% reduction in the incidence of labeled TDL suggesting that some virus eluted spontaneously. When 125I-virus-treated TDL were incubated in syngeneic serum, substantial amounts of virus eluted reducing the incidence of labeled cells by 50%. In addition, evidence was obtained by indirect immunofluorescence that syngeneic serum contains antibodies which react with sites on the lymphocyte surface exposed by the viral neuraminidase.
Hepatitis a Antigen Isolated from Liver and Stool: Immunologic Comparison of Antisera Prepared in Guinea Pigs
Authors: Dienstag Jules L. , Schulman Alan N. , Gerety Robert J. , Hoofnagle Jay H. , Lorenz Douglas E. , Purcell Robert H. , Barker Lewellys F.
Published: December 2022

Morphologically similar hepatitis A antigen particles (HA Ag)3 have been detected in the stools of patients with type A hepatitis and in the livers of marmosets experimentally infected with hepatitis A virus. To investigate the humoral antibody responses to these antigens and to compare the immunologic properties of HA Ag from these two sources, we immunized guinea pigs with either marmoset liver-derived HA Ag or with human stool-derived HA Ag in complete Freund's adjuvant and measured their antibody responses by immune electron microscopy (IEM) and immune adherence hemagglutination (IAHA). Antibodies reacting with both hepatitis A antigens were elicited in both groups. As determined by IEM, no distinction was seen between the reaction of guinea pig antiserum to each HA Ag tested under code when reacted against either liver-derived or stool-derived HA Ag. Antibodies elicited to marmoset liver-derived HA Ag and human stool-derived HA Ag had similar end point dilution titers by IAHA when tested against either “light” density (1.34 g/cm3) or “heavy” density (1.40 g/cm3) stool-derived HA Ag or liver-derived HA Ag. Low levels of antibody to normal liver or stool control antigens were observed transiently but did not obscure the specific response to HA Ag. These data suggest that morphologically similar HA Ag particles from different sources and with different densities are immunologically similar and may be identical. In contrast to the heterogeneity of surface antigens of hepatitis B virus, the comparable immunogenicity and apparent antigenic homogeneity of HA Ags derived from various sources may simplify the approach to development of a vaccine against viral hepatitis, type A.
Herpes Simplex-Raji(A44), a New Cell Line for Serologic Testing by Immunofluorescence
Authors: Seigneurin Jean-Marie , Desgranges Claude , Lavoue Marie-France , de-The Guy
Published: December 2022

An IF technique for the detection of HSV antibodies by using a lymphoblastoid cell line, Raji(A44), which continuously produce a constant amount of HSV antigen, is described. If and IH tests were found to be similar with respect to sensitivity and reproducibility. Sero-conversions detected by the CF test were detected with this cell line. The Raji(A44) line (cells in suspension) can be routinely used for the diagnosis of HSV infection.
Humoral Immunostimulation
Authors: Shearer William T. , Atkinson John P. , Parker Charles W.
Published: December 2022

When L cells were treated with anti-L cell antibody in medium depleted of complement, rapid increases in calcium uptake were obtained over a wide range of antiserum concentrations. Concomitant cell growth and viability studies demonstrated that stimulation of cell growth occurred at higher dilutions of antiserum whereas cytotoxicity occurred at lower dilutions. The stimulatory and toxic effects of antibody on cell growth were potentiated by complement as was the enhancement in calcium uptake. Sera deficient in C1R, C2,4D, C4, C3–C9 did not increase the calcium uptake response to antibody whereas augmentation did occur with C6-deficient serum. A specific role for complement was further indicated by the ability of purified complement components to restore the response to complement in complement-deficient sera. C3 with C3–C9 deficient serum, but not C2, C5, and C6 with C3–C9 deficient serum restored augmentation effects. Taken together with the results of previous studies it is apparent that complement augments both calcium and nucleoside uptake and that the effect is primarily via the classical complement pathway through C3.

Substrate saturation studies demonstrated that antibody activated the facilitated diffusion of calcium altering the Vmax but not the Km of transport whereas adddition of complement altered both the Vmax and Km. These findings suggest that one of the early effects of enhancing antibody upon tumor cell metabolism in vitro is to stimulate uptake of calcium. In view of the suspected role of Ca++ in cell proliferation the increase in cell-associated calcium may be important in the subsequent proliferative response.
The Primary Structure of Porcine C3a Anaphylatoxin
Authors: Corbin Neal C. , Hugli Tony E.
Published: December 2022

Porcine C3a was generated in whole porcine serum by inulin activation of enzymes of the alternative complement pathway. The C3a anaphylatoxin was isolated according to the procedures previously described by Hugli. The complete amino acid sequence for porcine C3a was determined utilizing automatic sequencing techniques in addition to manual subtractive Edman degradation and carboxypeptidase A, B, or Y digestion of isolated peptides. Porcine C3a is composed of a polypeptide chain containing 77 amino acid residues and has a m.w. of approximately 9,000 daltons. This C3a molecule is devoid of threonine, tryptophan, and carbohydrates. The proposed primary structure for porcine C3a is as follows:

Comparisons between the amino acid sequences of human and porcine C3a reveal that the six half-cystinyl and five aromatic residue positions are conserved. Conservation of these six half-cystinyl residue positions suggest that the disulfide arrangement remains identical in both anaphylatoxin molecules. Maintenance of three interconnected disulfide linkages helps to explain a near identity between the secondary structures of human and porcine C3a as indicated by circular dichroism measurements.

Particular attention was focused on the COOH-terminal region of the anaphylatoxins since an arginyl residue at position 77 is functionally essential in both human and porcine C3a. Five residue positions at the carboxy termini were conserved in both C3a molecules, and the sequence Leu-Gly-Leu-Ala-Arg probably relates directly to anaphylatoxin activity. A total of 23 residue replacements occur between human and porcine C3a which accounts for a 30% difference in primary structure. Although the C3a molecules exhibit identical biologic activity, this rather large structural difference readily explains the absence of a detectable immunologic cross-reactivity.
Virus-Enhanced Modulation of Cell Surface Antigens: Effect on Immune Lytic Susceptibility
Authors: Catanzaro Philip J. , Brandt Walter E. , Hogrefe Wayne R. , Phillips Stanley M. , Top Franklin H.
Published: December 2022

Monkey kidney cells, upon progressive subculture, became refractory to complement (C)-dependent immune cytolysis by anti-cell serum. Arbovirus infection restored these cells to a state of lytic susceptibility. Similar results were also obtained with antibody-dependent cellular cytotoxicity (ADCC), which is C independent. Antibodies raised against different subcultures varied considerably in lytic efficiency, indicating changing patterns of host cell expression during continuous subculture. Taken together with the fact that arbovirus infection restored the lytic efficiency of all antibody preparations to the same degree suggested some form of host cell antigen re-expression as a mechanism.

The results obtained in several exploratory experiments indicated that the antigenic re-expression responsible for the restoration of lysis was probably a local or selective rather than a generalized phenomenon. Thus, the amount of host cell surface antigen, measured by the use of mouse anti-cell serum and 125I anti-mouse globulin, was identical in both uninfected lytic susceptible and refractory cells, and decreased in both functional states following infection. Further, the binding of 125I concanavalin A, used to quantify surface glycoproteins, was similar in both lytic refractory and susceptible cells, and in both cases declined following virus infection. This result was incompatible with gross “masking” of cell surface antigens by exuberant production of surface coat material in lytic resistant cells. Finally, brief trypsinization of lytic resistant cells yielded an 8-fold increase in immune lysis, a result further consistent with local rather than generalized surface changes.

The data were discussed in terms of modulation of cell surface antigens affected both by repeated subculture and arboviral infection, and as a possible in vitro correlate of altered self-reactivity.
Inhibition of Normal Allogenic Lymphocyte Mitogenesis by a Soluble Inhibitor Extracted from Human Colonic Carcinoma
Authors: Remacle-Bonnet Maryse M. , Pommier Gilbert J. , Kaplanski Solange , Rance Roselyne J. , Depieds Raymond C.
Published: December 2022

Soluble extracts from human colonic tumors (STE) and from their hepatic metastases (SHME) were found to be unable to induce a proliferative response among normal allogenic lymphocytes. However, addition of these tissue extracts to cultures stimulated with various mitogens resulted in an almost complete inhibition of lymphocyte DNA synthesis. Nevertheless, they did not reduce the unstimulated lymphocyte spontaneous proliferation. Control experiments have shown that normal or nonmalignant tissues do not affect the lymphocyte reactivity to mitogens. The nonspecific immunosuppressive effect was found to be irreversible and to block lymphocyte activation at an early stage. The inhibitor was soluble (not sedimented at 220,000 × G for 2 hr) and not nonspecifically cytotoxic. STE was also found to induce morphologic alterations resulting in blastlike cell production. However, no mitotic figures were seen, even after colchicin treatment. It is suggested that STE might contain molecular component(s) which would exert a double effect: 1) trigger metabolic alterations responsible for the blast-like cell induction, and 2) inhibit the lymphoproliferative response. The significance of such a mechanism is discussed in connection with the nonspecific immunosuppression caused by a tumor and the immune unresponsiveness against the tumor itself. A preliminary characterization of this tumor material has shown that its molecular weight was about 70,000 and that it is not related to carcinoembryonic antigen or α-fetoprotein.
Xenogenization of Rat Erythroid Cells by Lymphatic Leukemia Virus: Its Role in Induction of Autoimmune Hemolytic Anemia
Authors: Kuzumaki Noboru , Moriuchi Tetsuya , Kodama Takao , Kobayashi Hiroshi
Published: December 2022

Newborn rats given injections of low doses of Friend lymphatic leukemia virus subsequently developed hemolytic anemia characterized by production of antierythrocyte autoantibody. Electron microscopy showed C-type virus particles budding from the cell membrane not only of lymphoid cells but also of erythrocyte precursor cells in bone marrow and spleen, suggesting that the erythroid cells were infected by the virus. In addition, erythrocyte precursor cells expressed virus-induced cell surface antigens detected by cytotoxicity tests. Normal syngeneic rats preimmunized with a Friend lymphatic leukemia virus-induced tumor and subsequently inoculated with bone marrow, spleen cells, or reticulocyte-rich fraction derived from other rats injected with high doses of the virus at birth produced cytotoxic antibody to the virus-induced tumor and antierythrocyte autoantibody. In contrast, rats subsequently inoculated with virus-infected thymus or lymph node cells produced cytotoxic antibody but not antierythrocyte autoantibody. These results indicate that “xenogenization,” previously shown for tumor cells and normal lymphoid cells, is also observed for rat erythroid cells and, further, that xenogenization of erythroid cells by Friend lymphatic leukemia virus is one of the most important factors in induction of autoimmune hemolytic anemia.
Activation of the Alternative Complement Pathway by Water-Insoluble Glucans of Streptococcus Mutans: The Relation between Their Chemical Structures and Activating Potencies
Authors: Inai Shinya , Nagaki Kazuyoshi , Ebisu Shigeyuki , Kato Keijiro , Kotani Shozo , Misaki Akira
Published: December 2022

Water insoluble, sticky glucan was synthesized by reacting glucosyltransferase obtained from the culture filtrate of cariogenic Streptococcus mutans OMZ 176 on sucrose. This glucan (OMZ 176 glucan), that consisted of the backbone chains of consecutive α-1,3 glucosidic linkages (65%) and the side chain of α-1,6 glucosidic bonds (35%), showed an ability to activate the alternative pathway of the complement system in human serum. Water soluble glucans synthesized from the reaction of glycosyltransferases of S. mutans strain AHT, FA-I, and Ingbritt with sucrose, that have a high content of α-1,6 and a low content of α-1,3 glucosidic linkages, were less effective to activate the complement system. Sephadex (G-25) and Dextran (T-2,000 and T-40), that consisted exclusively of α-1,6 glucosidic linkages, did not significantly activate the complement system. Controlled Smith degradation products of OMZ 176 glucan, composed of the α-1,3 linked glucose backbone chain alone, lost its activity. From these results it was concluded that both backbone chain of α-1,3 glucose units and branched α-1,6 glucose units were essential for the OMZ 176 glucan to activate the alternative pathway of the human complement system.
Large-Scale Isolation of Dane Particles from Plasma Containing Hepatitis B Antigen and Demonstration of a Circular Double-Stranded DNA Molecule Extruding Directly from Their Cores
Authors: Takahashi Takashi , Nakagawa Susumu , Hashimoto Takashi , Takahashi Kazuaki , Imai Mitsunobu , Miyakawa Yuzo , Mayumi Makoto
Published: December 2022

A method was developed to isolate, on a large scale, Dane particles from five liters of pooled plasma of asymptomatic carriers of hepatitis B antigen. It involved three successive ultracentrifugation procedures; the final preparation was more than 98% pure in Dane particles. Purified Dane particles had the density of 1.23 to 1.24 g/cm3. The concentration of nucleic acids in the preparation containing purified Dane particles was too low to be detected either by chemical or by spectrophotometric method. However, a circular double-stranded DNA molecule extruding directly from the core of Dane particles was clearly demonstrated by elelctron microscopic observations. The cores of Dane particles (hepatitis B core antigen; HBcAg) were prepared by treating the Dane particle preparation with mercaptoethanol and Nonidet P-40. They were sufficient both in purity and amounts to allow the determination of antibody to HBcAg by an immune adherence hemagglutination method.
Characterization of Lymphocyte Inhibition by Supernatants of Crowded Lymphocytoblasts
Authors: Green Stanley S. , Wistar Richard
Published: December 2022

Crowded lymphocytoblasts produce a factor (or factors) that inhibits the response of peripheral blood lymphocytes to mitogens. This factor is destroyed by pepsin in 1 hr but not by neuraminidase. No activity was lost when the factor was incubated with packed human red blood cells, human lymphocytes, mouse spleen cells, or tissue culture cells. An antiserum was made to the factor which was capable of partially inhibiting its activity. The factor is active at about 25 µg/ml of protein. Maximal inhibition occurs if the factor is present for the duration of the culture period. This factor may play a regulatory role in the immune response.
Schistosoma Mansoni Soluble Egg Antigens
Authors: Pelley Ronald P. , Pelley Robert J. , Hamburger Joseph , Peters Pierre A. , Warren Kenneth S.
Published: December 2022

A cell-mediated immunologic granulomatous response to Schistosoma mansoni eggs is now generally accepted as being responsible for the hepatosplenic disease of chronic schistosomiasis. Previous investigations have demonstrated that a soluble extract of S. mansoni eggs (SEA) both induces and elicits granulomatous hypersensitivity and other forms of cell-mediated immunologic reactivity. Mice with chronic light S. mansoni infections show spontaneous suppression of granulomatous hypersensitivity in the presence of high levels of anti-SEA antibodies. Immunodiffusion analysis using antiserum obtained from these mice and SEA resulted in the identification of three major serologic antigens which have been designated MSA1, MSA2, and MSA3. Initial studies with Sephadex G-200 gel filtration and polyacrylamide gel electrophoresis indicated that the three antigens were markedly different in m.w. and at least two of the three were glycoproteins. The antigens were then extracted from crude SEA by adsorption to a concanavalin A Sepharose affinity column. The eluted antigens were separated from each other by ion exchange chromatography on DEAE cellulose. On polyacrylamide gel electrophoresis (PAGE) MSA1 and MSA2 were homogenous; MSA3 was estimated to be 70% pure. The purified antigens were radiolabeled and were passed through Sephadex G-200 or Bio-Gel A 1.5 columns to determine their m.w. These studies have shown that MSA1 and MSA2 are glycoproteins of m.w. 137,000 and 465,000 daltons, respectively; the m.w. of MSA3 is in the range of 50,000 to 70,000 daltons. PAGE of the purified antigens revealed the following R.F.s: MSA1 = 0.34, MSA2 = 0.20, and MSA3 = 0.48. MSA1 and MSA2 were employed in radioimmunoassays using the ammonium sulfate method of Farr. On the basis of immunodiffusion analysis and radioimmunoassay, MSA1 exhibits a degree of stage and species specificity consistent with the granulomatous response to S. mansoni eggs. The potential specificity of the MSA1 radioimmunoassay and its great sensitivity suggest a role in the immunodiagnosis of schistosomiasis.
Suppression of in Vitro Lymphocyte Stimulation in Mice by Uterine and Placental Extracts
Authors: Kouttab Nicola M. , Fowler Arnold K. , Strickland James E. , Hellman Alfred
Published: December 2022

The effects of uterine extracts from virgin mice and uterine and placental extracts from mice at various stages of gestation on lymphocyte stimulation in vitro were compared. Placental and uterine extracts from mice in gestation, at 1% final concentration by volume, caused marked depression of lymphocyte reactivity to phytohemagglutinin-P (PHA) and to allogeneic cells in two-way mixed leukocyte cultures (MLC). An equivalent concentration of uterine extract from virgin mice caused slight depression of lymphocyte reactivity. Suppression was most marked with uterine extracts of mice at 15 days of gestation. At least two factors of different m.w. in uterine and placental extracts were responsible for suppression of lymphocyte reactivity. The higher m.w. fraction, present in small amounts in the uteri of virgin mice, was markedly increased in uteri of pregnant mice. This fraction contained immunoglobulin G (IgG). These factors may be involved in a protective role for the fetus from rejection by the mother as an allograft, thus assuring successful pregnancy.
Chemotaxis of Human Polymorphonuclear Neutrophils under Agarose: Morphologic Changes Associated with the Chemotactic Response
Authors: Nelson Robert D. , Fiegel Vance D. , Simmons Richard L.
Published: December 2022

Studies of morphologic changes of leukocytes responding to a chemotaxin gradient have been limited due, in part, to difficulties encountered with detailed electron microscopic analysis of cells migrating through membrane filters or on glass slides. The recently developed migration under agarose method provides a new opportunity to view and compare morphologies of cells migrating spontaneously and in response to a chemotaxin gradient. The present report describes the morphologic features we have observed by scanning electron microscopy of human polymorphonuclear leukocytes both spreading on a cover slip and migrating under agarose. We demonstrate that cells migrating under agarose exhibit a morphology that differs distinctly from that of cells spreading on a cover slip and, further, that the cytoplasmic periphery of cells responding to a chemotactic gradient under agarose becomes very irregular due to the formation of blebs or small pseudopodium-like structures.
Hapten-Sandwich Labeling
Authors: Cammisuli Salvatore , Wofsy Leon
Published: December 2022

Bifunctional reagents have been prepared which permit extensive coupling of haptens to antibodies with retention of antibody-binding capacity. This modification procedure increases the sensitivity with which cell-surface antigens can be labeled for fluorescent or electron microscopy by the hapten-sandwich method to a level comparable to or greater than may usually be achieved by conventional indirect techniques. With two different haptens, and amplifying with anti-hapten antibodies bearing fluorescent or other markers, different antigens can be distinguished readily on a single cell or on separate cells. Mouse alloantigens detected only with difficulty by conventional fluorescence can be discerned clearly and with high specificity.

The reagent, either methyl hydroxybenzimidate (HB) or methyl 3,5-dihydroxybenzimidate (DHB), is first reacted with a diazonium phenyl hapten. Since the resulting azoderivative retains the imido ester function, the reaction solution can be used directly to amidinate antibodies. Both HB and DHB are easily prepared and may be stored for use as needed. With these reagents, the hapten-sandwich procedure may be applied to proteins other than immunoglobulins, e.g., hormones and mitogens. In addition to labeling antigens for fluorescent or electron microscopy, haptenamidinated antibodies or other ligands may be used in conjunction with appropriately modified antihapten amplifiers for a variety of purposes in cell biology, such as radioisotope studies, selective cell killing or suppression, and isolation of membrane antigens.
Antibody Response to Lymphoma Antigens in Syngeneic and H-2 Compatible Mice
Authors: Dalton James G. , Fujimoto Shigeyoshi , Sehon Alec H.
Published: December 2022

The antibody response to immunization with A/J lymphoma L1117 cells by syngeneic A/J mice and by H-2 compatible B10. A mice has been shown to be highly specific for tumor antigens of the lymphoma cells. In both strains the response exhibits a very slow rise in cytotoxic antibody levels reaching a maximum only after 3 months of biweekly injections. It is also unusual in that cytotoxic activity is primarily due to IgM antibodies even after 20 weeks of immunization. The similarity between these characteristics and those reported for “T-independent” antigens suggests that tumor antigens on L1117 cells may be unable to stimulate helper T cells.
Effect of α-Fetoprotein and Other Serum Factors Derived from Hepatoma-Bearing Rats on the Mixed Lymphocyte Response
Authors: Parmely Michael J. , Thompson John S.
Published: December 2022

Serum α-globulin fractions isolated by physiocochemical techniques from normal adult Buffalo rats suppressed lymphocyte proliferation in vitro. The factors responsible for mixed lymphocyte culture suppression appeared to be strain specific since they were not demonstrable in the same fractions from normal LBN rat serum. Similar fractionation of the serum from Buffalo rats bearing the Morris Hepatoma 7777 obtained from two different sources also yielded suppressive protein fractions that differed both chemically and functionally.

Both variants of this hepatoma produced high serum concentrations of α-fetoprotein (AFP), providing an opportunity to study the possible immunoregulatory role of their fetal-associated globulins. Fractions rich in AFP that lacked other serum α-globulins were obtained by gel filtration chromatography and were devoid of any in vitro immunosuppressive activity. When AFP that was further purified by immunoabsorption was added to mixed lymphocyte cultures, no effect was observed at doses below 400 µg/ml. The MLC response was augmented with higher doses, similar to albumin purified by the same methods.
Reduction of the in Vitro Cytotoxic Lymphocyte Response Produced by in Vivo Exposure to Semiallogeneic Cells: Recruitment or Active Suppression?
Authors: Miller Richard G. , Phillips Robert A.
Published: December 2022

Twenty-four hours after intravenous injection of (C57BL/6 × DBA/2) F1 hybrid lymphoid cells into C57BL/6 mice, the in vitro cytotoxic lymphocyte (CL) response of the C57BL/6 spleen and LN cells against DBA/2 alloantigens can be greatly reduced. The reduction is specific for the foreign alloantigens injected; is reduced by irradiating the cells before injection; develops significantly within 10 min of injection, thereafter increasing with a time constant of about 2 hr; varies with the number and origin of cells injected; can be induced by several subpopulations of injected cells within one tissue; varies with the host organ tested (e.g., injection of 104 semiallogeneic LN cells can reduce hose LN response more than 100-fold and have no effect on host spleen); and (for the LN response) is largely reversed by treatment with anti-H-2 antibody plus complement specific for the injected cells. The reduction appears to be induced by grafted cells entering host lymphoid organs rather than, e.g., host cells being specifically recruited to go elsewhere.

When cocultured with normal cells, lymphoid cells from mice injected as above do not reduce the in vitro CL response of the normal cells but do if left in vivo for 7 days before testing. If left 1 day in vivo, followed by 4 days culture in vitro with the same alloantigen, they strongly reduce the normal cell response. The reduction is increased by both the in vivo and in vitro exposure. The grafted cells, once in the host lymphoid tissue, appear to activate a host suppressor mechanism which develops in vitro and reduces the CL response.
Two Types of Antibody Dependent Cell-Mediated Cytotoxicity, Arming and Sensitization
Authors: Imir T. , Saksela Eero , Mäkelä O.
Published: December 2022

Haptenated chicken erythrocytes, anti-hapten antibodies and normal spleen cells were used for a study of parameters of antibody-dependent cell-mediated cytotoxicity (ADCC). Anti-hapten antibody was introduced to the reaction either bound to the target cells (sensitization) or to the effector cells (arming). Our data suggest that the same effector cells and the same antibodies were responsible for both types of reactions. More antibody was required for arming (ca. 1 ng/ml) than for sensitization (ca. 10 pg/ml).

Antibodies of several mammalian species could cooperate in ADCC with human, rat, or guinea pig spleen effector cells, but fowl antibodies were inefficient in the arming of mammalian effector cells.

Rat and human spleen cells were powerful effector cells whereas guinea pig, rabbit, and mouse spleens yielded weakly cytotoxic suspensions. Rabbit antibodies, on the other hand, were efficient, but rat antibodies were less efficient in ADCC. Mouse and guinea pig antibodies appear to take an intermediate position.

Our data suggest that ADCC effector cells of the spleen contain phagocytic and nonphagocytic cells, and the non-phagocytic effector cells still constitute a heterogeneous population.
Isolation of a Human Lymphocyte Mitogen from Wheat Germ with N-Acetyl-D-Glucosamine Specificity
Authors: Brown John M. , Leon Myron A. , Lightbody James J.
Published: December 2022

A lectin, isolated from wheat germ by affinity chromatography on chitin, was mitogenic for purified human peripheral blood lymphocytes. Peak incorporation of 3H-thymidine was observed after incubation of lymphocyte cultures with wheat germ mitogen for 7 to 10 days. When lymphocytes were separated into two fractions based on their ability to form rosettes with unsensitized sheep erythrocytes, the mitogen induced a negligible proliferative response in either fraction. Mixing experiments demonstrated a strong response in the T lymphocyte fraction which required the collaboration, but not proliferation, of cells present in the nonrosetting fraction. Stimulation was specifically abolished by addition of N-acetyl-D-glucosamine at initiation of culture.
Cytomegalovirus Specific IgM and IgG Response in Humans Studied by Radioimmunoassay
Authors: Knez Violeta , Stewart John A. , Ziegler Donald W.
Published: December 2022

An indirect solid phase micro-radioimmunoassay (RIA) was adapted for the measurement of anti-cytomegalovirus class-specific immunoglobulin M (IgM) and immunoglobulin G (IgG). Cytomegalovirus (CMV) antigen (Ag) was added to the wells of microtiter plates and desiccated onto the bottom surface of the wells. Serial dilutions of human CMV antisera were added and allowed to react with the Ag. The amount of viral antibody (Ab) was determined by measuring the specific binding of 125I-labeled goat antihuman IgG (125I-AHIgG) and/or IgM (125I-AHIgM). The multiple factors which influence the test were determined.

Serial samples of sera from CMV-positive patients showed progressive increases in Ab titers on the basis of specific binding of 125I-AHIgG. The titers of IgM class anti-CMV Ab were also determined with the same sera, and enhancement of the titers was demonstrated when the incubation periods of the first Ag-Ab reaction were extended from 1 to 3 hr.

The competition between the different Ig classes of Ab for CMV Ag and the involvement of rheumatoid factor (RF) were investigated. The anti-CMV IgG that may compete with the IgM determination was removed by adsorption of sera with Staphylococcus aureus (Cowan 1). The RF that would cause false anti-CMV IgM results was adsorbed with glutaraldehyde-insolubilized IgG. Our results indicate that this RIA is a practical, specific, and reproducible technique for detection of specific IgG and IgM Ab to CMV.
Trypsin-Induced Increase in Intracellular Cyclic AMP of Lymphocytes
Authors: Shneyour Amir , Patt Yehuda , Trainin Nathan
Published: December 2022

Trypsin increases intracellular levels of cyclic AMP (cAMP) in lymphocytes. The trypsin-induced increase in cAMP is blocked by specific trypsin inhibitors and by high concentrations of different proteins. Several proteolytic enzymes from various sources, including other pancreatic proteases, do not cause an increase in cAMP under the same experimental conditions. Immobilized trypsin induces the same increase in cAMP as does free trypsin. The trypsin-induced rise in cAMP is not due to inhibition of cAMP phosphodiesterase, but consistent activation of adenylate cyclase by trypsin could not be demonstrated. The extent of the trypsin-induced increase in intracellular cAMP correlates with the type of the lymphocyte and with the state of maturity attained by the cells. Transformed lymphocytes and nonlymphoid cells do not react at all.
Kinetic Analysis of Target Cell Destruction by Effector T Cells
Authors: Thorn Richard M. , Henney Christopher S.
Published: December 2022

Murine cytotoxic T cell activity was evaluated in analogy with the methods used to characterize enzymes. Concordant with this analogy, the relationship between “steady-state” lytic rates and target cell (“substrate”) number was described by a hyperbola, the asymptote of which was the maximum rate of lysis (“V”) and was directly proportional to the number of killer cells. The number of target cells required to reach a rate of one-half V (“K”) was independent of the number of effector cells, but was found to vary with the total cell concentration in the cultures.

The kinetic analogy was extended to analyze this variability by studying the inhibition of cytolysis caused by normal syngeneic lymphocytes. A value for K in the absence of non-killer cells (“Kh”) was evaluated. This term, which was independent of effector cell frequency, was a measure of lytic efficiency and, as such, was used to compare various effector cell populations.

The Kh value for a number of lymphoid cell populations from identically immunized C57BL/6 mice was found to vary considerably, thereby providing direct evidence that effector populations have different lytic efficiencies. Because of this variability, the widely used practice of comparing killer cell populations solely on the basis of their relative lytic activity may often be invalid.
Concurrently Circulating Hepatitis B Surface Antigen and Heterotypic Anti-HBs Antibody
Authors: Le Bouvier George L. , Capper Roy A. , Williams Alan E. , Pelletier Marie , Katz Alfred J.
Published: December 2022

In the early days of testing for hepatitis B surface antigen (HB8Ag)2 and the homologous antibody (anti-HB8), there were reports of antigen and antibody co-existing in the same serum specimen (1, 2). Their subspecificities were not determined; but their homotypic character was evidently inferred, and in keeping with this idea were certain results obtained by complement fixation (3), and electronmicroscopic observations of circulating complexes apparently made up of HB8Ag-positive Australia particles and antibody molecules (4). Some recent studies have adduced further evidence for the existence of such complexes (5), whereas others have failed to substantiate the concurrent presence of HB8Ag and homologous anti-HB8, at least in the blood of asymptomatic carriers of the antigen (6).

With the definition of distinct antigenic phenotypes of HB8Ag, there arose the theoretical possibility that Australia particles of one phenotype might co-circulate with free anti-HB8 antibody directed against antigenic configurations characteristic of a different phenotype.
Studies on the Mechanism of Lymphocyte-Mediated Cytolysis
Authors: Rubens Robert P. , Henney Christopher S.
Published: December 2022

Alloimmune spleen cells (C57BL/6 anti P815), but not normal spleen cells, lyse syngeneic (EL4) target cells in the presence of Con A. Con A dependent cytotoxicity was mediated by T cells and required the continued presence of lectin. Cytolysis in the presence of a succinylated derivative was equivalent to that seen with the parent Con A molecule.

In contrast to previous reports of Con A dependent cytolysis, however, we conclude that lysis is not primarily caused by directly cytotoxic T cells. The reasons for this conclusion are: Removal of directly cytotoxic cells by adsorption on P815 monolayers did not alter the Con A dependent cytolysis of EL4 cells;Populations in which no direct T killers were demonstrable (e.g., spleen cells harvested 5 days after alloimmunization) lysed both P815 and EL4 cells in the presence of Con A; andCon A dependent cytolysis, but not direct cytotoxicity, could be induced by culturing normal C57BL/6 spleen cells for 4 days with a sonicated extract of P815 cells.

We hypothesize that the cell “activated” to lyse targets in the presence of Con A is a T cell which has differentiated lytic potential following alloantigenic stimulation, but has either insufficient density or affinity of antigen receptors to serve as a directly cytotoxic cell. The role of Con A is viewed as 2-fold: i) to “bridge” killer and target cell, and ii) to “activate” the effector.
Development of Mast Cells in Vitro
Authors: Ishizaka Teruko , Adachi Toshihiro , Chang Tong-Hsuan , Ishizaka Kimishige
Published: December 2022

Mast cells were obtained by long term culture of rat thymus cells on rat embryonic fibroblast monolayers. Pure mast cell preparations obtained by culture were incubated with 125I-labeled rat E myeloma protein to study receptors for IgE on their surface. When the cells were obtained after 35 to 45 days culture, the average number of receptors per mast cell was 100,000 to 400,000. An equilibrium constant of the binding reaction between their receptor and rat IgE was in the order of 108 M-1. The histamine content of the cultured mast cells was 0.2 to 5 µg/106 cells. The measurement of histamine content in mast cells recovered after different periods of culture suggested that the histamine content increased with maturation. Even after 45 to 50 days culture, the histamine content of cultured mast cells was significantly lower than that in rat peritoneal mast cells. The cultured mast cells were passively sensitized in vitro with rat IgE antibody against Nippostrongylus brasiliensis. The sensitized cells released histamine upon incubation with the antigen. It was also found that cultured mast cells released histamine upon exposure to compound 48/80. These results indicated that cultured mast cells have physiologic functions similar to those of normal rat mast cells, but they have not reached full maturation.
Delineation and Quantitation of Human Peripheral Blood Lymphocyte Subpopulations by Electrophoretic Mobility and Role of Surface Charge in Cell to Cell Interaction
Authors: Hanjan S. N. S. , Talwar G. P. , Kidwai Zeba , Nath Indira
Published: December 2022

A modified procedure is proposed for determination of electrophoretic mobility (EPM) of lymphocytes which enables readings on 200 cells from a sample size of 5 × 106 lymphocytes. In normal human peripheral blood, B lymphocytes (bearing surface immunoglobulin and receptor for activated C3) were found to carry the lowest charge (0.94 ± 0.05 µ/sec/V/cm). Their number on the EPM basis was 16.9 ± 4.2%. Two subpopulations of T lymphocytes, one carrying high charge (TH) with a mean EPM of 1.26 ± 0.04 µ/sec/V/cm and the other carrying lower charge (TL) with a mean EPM of 1.11 ± 0.03 µ/sec/V/cm were discernible. The former subpopulation of T cells formed spontaneous rosettes with SRBC in 10 min and the latter represented the remaining T lymphocytes which formed rosettes with 24 hr of incubation with SRBC.

The number of early rosette-forming cells increased from 69 to 89% when T cells were incubated with neuraminidase-treated SRBC. These observations suggest that complementary electrostatic charge on T cells and SRBC are among the forces facilitating formation of rosettes between these cells.
Lack of Interaction of Circulating T Cells with Phytohemagglutinin in Bacillary Positive Untreated Lepromatous Leprosy Patients—Identification of Subpopulation of Lymphocytes by Shifts in Electrophoretic Mobility
Authors: Talwar G. P. , Hanjan S. N. S. , Mehra V. L. , Kidwai Zeba
Published: December 2022

Incubation of human peripheral blood lymphocytes from normal healthy subjects with phytohemagglutinin (PHA), causes the reduction of the surface charge of a subpopulation of T cells by 1363 ± 242 e.s.u./cm2. The affected subpopulation was predominantly the high charge-bearing cells identifiable with early (10 min) rosette-forming cells with sheep erythrocytes. Purified lymphocytes obtained from untreated bacillary-positive, lepromatous leprosy patients contained high charge-bearing T lymphocyte subpopulation. However, incubation with PHA did not result in the shift of electrophoretic mobility of these cells, suggesting the absence of interacting sites for the mitogen on the surface of these cells. The absence of mitogen-interacting sites is not an inherent trait of leprosy patients; the surface charge of lymphocytes from Dapsone-treated bacillary-negative subjects was reduced upon incubation with PHA. A close correlation was found between the number of cells whose charge alters on incubation with PHA and the transformation index obtained with this mitogen.