Appearance of T lymphocyte-derived proteins specific for the immunizing antigen in serum during a humoral immune response.

Some T lymphocytes produce extracellular proteins that bind nominal antigen specifically (TABM), and these proteins exhibit potent immunoregulatory activity. We have utilized an ELISA for Ag binding by Ag-specific TABM to detect and quantitate the appearance of Ag-specific TABM in murine serum during a humoral Ir to protein Ag. The TABM response was specific for the inducing Ag, stronger and more rapid during a secondary response, and temporally distinct from the appearance of Ig. The non-Ig serum TABM were bound by mAb specific for TCR-alpha chains and isolated by affinity for Ag were Mr 110,000 polypeptides. The TABM response did not occur in scid/scid mice unless the mice were reconstituted with thymocytes and thymocyte-reconstituted scid/scid mice produced TABM, but did not produce Ig. The results suggest that soluble TABM are an Ag-specific humoral manifestation of the Ir of some T lymphocytes.     

Ontogeny and expression of non-MHC-restricted T cell antigen-binding molecules by thymocytes and peripheral T cell subsets

The ontogeny of T cells which express major histocompatibility complex (MHC)-unrestricted T cell antigen-binding molecules (TABM) on the cell membrane was investigated. We used a rabbit anti-mouse TABM antiserum to investigate the expression of TABM by subsets of adult thymocytes, peripheral T cells, and thymocytes during gestation. TABM are expressed by CD4+, CD8-, CD4+, CD8+ thymocytes and single-positive thymocytes. During gestation, TABM are expressed as early as Day 16, and at birth the expression of TABM on thymocytes has reached adult levels. Data are also presented which suggest that the expression of membrane TABM (mTABM) on peripheral T cells can be upregulated during T cell activation. The results suggest that TABM are expressed by different T cell subsets and that TABM+ cells may utilize the same intrathymic developmental pathway as that of T cells which express the alpha/beta T cell receptor.     

Studies on the transfer of protective immunity with lymphoid cells from mice immune to malaria sporozoites.

In an effort to understand the mechanisms involved in the protective immunity to malarial sporozoites, an A/J mouse/Plasmodium berghei model was studied. Protective immunity could consistently be adoptively transferred only by using sublethal irradiation of recipients (500 R); a spleen equivalent (100 X 10(6))of donor cells from immune syngeneic mice; and a small booster immunization (1 X 10(4)) of recipients with irradiation-attenuated sporozoites. Recipient animals treated in this manner were protected from lethal challenge with 1 X 10(4) nonattenuated sporozoites. Immune and nonimmune serum and spleen cells from nonimmune animals did not protect recipient mice. Fewer immune spleen cells (50 X 10(6)) protected some recipients. In vitro treatment of immune spleen cells with anti-theta sera and complement abolished their ability to transfer protection. This preliminary study suggests that protective sporozoite immunity can be transferred with cells, and that it is T cell dependent.     

Immunoglobulin G is the main protective antibody in mouse vaginal secretions after vaginal immunization with attenuated herpes simplex virus type 2.

We investigated the protective role of antibodies in vaginal secretions of mice that were immune to vaginal challenge with herpes simplex virus type 2 (HSV-2). Unfractionated vaginal immunoglobulins from immune and nonimmune mice and affinity-purified immunoglobulin G (IgG) and secretory IgA (S-IgA) from immune secretions were adjusted to their concentrations in vivo. Wild-type HSV-2 was incubated in the immunoglobulin preparations for 15 min in vitro, followed by inoculation into vaginae of nonimmune mice. HSV-2 was neutralized by unfractionated antibody and purified IgG from immune secretions but not by unfractionated nonimmune antibody or by purified immune S-IgA. The protective effect of IgG in vivo was investigated by passively transferring purified serum IgG from immune and nonimmune donors to nonimmune recipients before vaginal challenge infection. Immune IgG significantly reduced the percentage of vaginal epithelium infected, concentrations of shed virus protein in the vaginal lumen, and illness scores, even though the viral antibody titers in serum and vaginal secretions of recipient mice at the time of challenge were only 29 and 8%, respectively, of those in actively immunized mice. Additionally, removal of vaginal secretions from immune mice 10 min before vaginal challenge with HSV-2 significantly increased the concentration of shed virus protein in the vaginal lumen after challenge. Collectively, the data indicate that IgG antibody in vaginal secretions of immune mice provides early protection against vaginal challenge infection, probably by neutralizing virus in the vaginal lumen. In contrast, S-IgA antibody contributed relatively little to immune protection of the vagina.     

Induction of tetravalent protective immunity against four dengue serotypes by the tandem domain III of the envelope protein

In the present study, the domain IIIs of all four dengue virus (DENV) serotypes were connected sequentially to construct the tandem domain III. The resulting DNA fragment was then cloned into pBAD/Topo ThioFusion plasmid. After induction, the Escherichia coli expression protein was purified and used to immunize BALB/c mice by subcutaneous route. The sera from mice immunized with the purified protein were confirmed to contain specific high antibody titers against DEN1, DEN2, and DEN4, and moderate antibody titer against DEN3. In suckling mouse model, 70% of the mice challenged with DEN1, DEN2, and DEN4 in combination with sera from mice immunized with the purified protein were protected, and 18% of the mice challenged with DEN3 in combination with the same sera were protected. Our data suggest that the tandem domain III of the envelope protein can be used as a potential tetravalent dengue vaccine based on a single antigen.     

Expression of non-MHC-restricted T cell antigen-binding molecules by thymic lymphocytes

Heterologous antisera which recognize non-major histocompatibility complex (MHC)-restricted T cell antigen-binding molecules (TABM) were used to characterize the expression and structure of TABM on thymic lymphocytes. Approximately 70% of thymocytes express membrane molecules bound by anti-TABM antibodies (mTABM). Antibody activity for thymocyte TABM could be removed by adsorption to splenic T cells, but not by adsorption to splenic B cells. Similarly, adsorption of the antiserum to thymocytes or splenic T cells removed antibody activity to a purified TABM whereas adsorption with B cells had no effect. Radioiodinated thymic and splenic T cell mTABM were resolved by 2D-polyacrylamide gel electrophoresis and when reduced, both populations of mTABM migrated primarily as Mr 23,000 proteins with an isoelectric point range of 6.8-7.8. Multimers of this protein were also observed at Mr 85-97,000 and 130-150,000 on both thymocytes and splenic T cells. These data indicate that MHC-unrestricted antigen-binding molecules are expressed by a majority of thymocytes and these thymic TABM are structurally and antigenically similar to mTABM on peripheral cells. This suggests an ontogenic relationship between thymic TABM and peripheral TABM.     

Separation of anti-histone antibodies from nonimmune histone-precipitating serum proteins, predominantly alpha2-macroglobulin.

Calf thymus histories formed precipitates with human, rabbit, guinea pig, mouse, goat, or sheep sera regardless of whether the subjects were immunized with histones. Their order of activity in precipitation was: H4 >H3 >H2a >H2b ? H1. Precipitation occurred with more than one serum protein fraction, but not with purified IgG, IgM, or crystallized albumin. The major nonimmune precipitant had the size and charge properties of α2-macroglobulin. Serological analysis with anti-α2-macroglobulin antiserum confirmed the presence of this protein in the precipitating fractions, and its selective depletion from histone-precipitated serum. Specific anti-histone antibodies were separated from the nonimmune precipitants by DEAE-cellulose chromatography.     

Serum opsonic activity in rodent malaria: functional and immunochemical characteristics in vitro.

The functional and immunochemical characteristics of serum opsonic activity in rodent malaria were examined in the present study. Schizont- and late trophozoite-enriched populations of Plasmodium berghei-infected red blood cells (IRBC) were isolated on a Ficoll density-gradient and used in an in vitro phagocytosis system composed of serum and monolayer cultures of rat peritoneal macrophages. Hyperimmune serum augmented the phagocytosis of IRBC to a greater degree than did nonimmune serum. When either IRBC or macrophages were pre-incubated with serum, the phagocytosis-promoting factors acted on the IRBC rather than on the macrophages in a manner characteristic of serum opsonins. The opsonic activity was specific for IRBC since noninfected red blood cells were rarely phagocytized and were unable to absorb opsonic activity from serum. The opsonic activity of both hyperimmune and nonimmune sera was heat stable, and unaffected by agents known to inactivate or inhibit complement (cobra venom factor and ethylenediaminetetraacetic acid). Finally, the opsonic activity was identified in preparations of purified IgG isolated from both hyperimmune and nonimmune sera.     

Macrophage immunity to influenza virus: in vitro and in vivo studies.

Using M-TUR, a macrophage-adapted avian influenza A virus (Hav1, Nav3), antiviral resistance of peritoneal macrophages obtained from specifically or nonspecifically immunized mice towards in vitro infection was assessed. M-TUR grew to high titers in macrophages from nonimmune mice thereby causing a marked cytopathic effect. In contrast, peritoneal macrophages from mice specifically immunized with TUR virus were not affected by infection with M-TUR in vitro. This antiviral immunity was specific: mice immunized with antigenetically unrelated influenza strains such as influenza A/Hong Kong/1/68 (H3, N2) or influenza B/Lee yielded susceptible macrophages. Specific macrophage immunity could be abrogated by trypsin treatment in vitro. Susceptible macrophages from nonimmune hosts became resistant following in vitro exposure to homologous anti-TUR sera. Peritoneal exudate cells from BCG-infected animals were less susceptible to in vitro challenge with M-TUR than control macrophages. In vivo treatment of mice with the unspecific immunostimulants BCG or Corynebacterium parvum did not protect the animals against lethal infection with a hepatotropic variant of TUR.     

Immunoproteasome expression in a nonimmune tissue,the ocular lens

Interferon gamma (IFN gamma) induces the expression of three catalytic subunits of the 20S proteasome that can replace their constitutive homologues to form the "immunoproteasome," named to reflect its antigen presentation function. However, immunoproteasome levels and their modulation in nonimmune tissues remain unknown. A disrupted lens differentiation program observed in transgenic mice that constitutively express IFN gamma in the immune-privileged lens tissue suggests a role for this cytokine in differentiation. We have developed a competitive RT-PCR assay that demonstrates substantially increased levels of immuno subunits and unchanged levels of constitutive subunits in transgenic compared to wild-type lenses. Similar results were observed with IFN gamma treated alpha TN4-1 lens epithelial cells. A comparison of these subunits in different immune and nonimmune mouse tissues revealed unique expression patterns. The presence of immuno subunits in nonimmune tissues such as lens suggests that the immunoproteasome may also have nonimmune functions, such as that in lens differentiation.     

Purification and characterization of distinct type of mannose-sensitive fimbriae from Salmonella typhimurium

Abstract The fimbriae (E₄) of a virulent strain of Salmonella typhimurium were purified by ion exchange chromatography in an FPLC system. They had a channelled appearance under transmission electron microscope and showed a major structural subunit of 17-kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The purified fimbriae were found to agglutinate guinea pig erythrocytes, but this effect was inhibited in presence of D-mannose. Immune sera raised against the Mono-Q purified fimbriae (E₄) showed cross-reactivity with the type-1 fimbriae (F₁) composed of 21-kDa fimbrin subunit, purified by a different method from the same strain.     

Association between anti-Sm and anti-ribosomal P protein autoantibodies in human systemic lupus erythematosus and MRL/lpr mice

Anti-Sm and anti-ribosomal P protein antibodies show a high degree of specificity for the disease SLE. To determine whether a relationship between these two autoantibodies existed, the frequency of anti-P was determined in sera with and without anti-Sm activity. Of sera from lupus patients with anti-Sm 18/65 (28%), and 6/55 (11%) of sera without anti-Sm had anti-P as determined by an ELISA using a recombinant P2-beta-galactosidase fusion protein as Ag (p less than 0.05). The levels of anti-P were significantly higher in sera containing anti-Sm (0.37 +/- 0.45) than in sera without anti-Sm antibodies (0.18 +/- 0.20) (p less than 0.01). Similarly, a significantly higher proportion of anti-P positivity was found in autoimmune MRL/Mp-lpr/lpr mice positive for anti-Sm (11/53 = 21%) compared to age- and sex-matched mice without anti-Sm (3/53 = 6%) (p less than 0.05). The IgG subclass distributions for anti-Sm and anti-P antibodies were similar in the MRL mice (IgG2a greater than IgG2b greater than IgG3 greater than IgG1). The association did not reflect polyclonal B cell activation in a proportion of MRL mice because no significant differences were observed in anti-DNA, antichromatin or total serum IgG levels in mice with and without anti-Sm or, in mice positive for both anti-P and anti-Sm compared to mice positive for anti-Sm alone. Cross-inhibition experiments excluded the possibility that the Sm and P protein Ag shared a common epitope. Longitudinal measurement of anti-P and anti-Sm antibody levels by ELISA in three mice indicated that both antibodies first appeared at about 3 to 4 mo of age and fluctuated two- to threefold over 3 to 8 mo with independent peaks of activity. Recent observations regarding a relationship between anti-Sm and autoantibodies to other ribosomal proteins suggest that the association may be explained by an immune response to epitopes coassociated on the ribosome.     

Induction of tolerance to syngeneic IgE in neonatal mice

We have previously shown that adult A/J mice are tolerant to syngeneic IgE at the level of T cells, but not B cells. T cells of mice are responsive until the age of 2 to 3 wk, which correlates with the time of appearance of serum IgE. Tolerance can be induced earlier by neonatal administration of IgE in saline. We report here that purified nonimmune adult B cells, but not T cells, can transfer the state of tolerance to neonatal mice. As few as 2 x 10(6) B cells are effective. If IgE-bearing or IgE-secreting cells prove to be responsible, the amount of cell-bound IgE that can induce tolerance must be very small. The results also indicate that suppressor T cells do not have a major role in maintenance of self-tolerance to IgE.     

Construction, Safety, and Immunogenicity in Nonhuman Primates of a Chimeric Yellow Fever-Dengue Virus Tetravalent Vaccine

We previously reported construction of a chimeric yellow fever-dengue type 2 virus (YF/DEN2) and determined its safety and protective efficacy in rhesus monkeys (F. Guirakhoo et al., J. Virol. 74:5477-5485, 2000). In this paper, we describe construction of three additional YF/DEN chimeras using premembrane (prM) and envelope (E) genes of wild-type (WT) clinical isolates: DEN1 (strain PUO359, isolated in 1980 in Thailand), DEN3 (strain PaH881/88, isolated in 1988 in Thailand), and DEN4 (strain 1228, isolated in 1978 in Indonesia). These chimeric viruses (YF/DEN1, YF/DEN3, and YF/DEN4) replicated to ~7.5 log(10) PFU/ml in Vero cells, were not neurovirulent in 3- to 4-week-old ICR mice inoculated by the intracerebral route, and were immunogenic in monkeys. All rhesus monkeys inoculated subcutaneously with one dose of these chimeric viruses (as monovalent or tetravalent formulation) developed viremia with magnitudes similar to that of the YF 17D vaccine strain (YF-VAX) but significantly lower than those of their parent WT viruses. Eight of nine monkeys inoculated with monovalent YF/DEN1 -3, or -4 vaccine and six of six monkeys inoculated with tetravalent YF/DEN1-4 vaccine seroconverted after a single dose. When monkeys were boosted with a tetravalent YF/DEN1-4 dose 6 months later, four of nine monkeys in the monovalent YF/DEN groups developed low levels of viremia, whereas no viremia was detected in any animals previously inoculated with either YF/DEN1-4 vaccine or WT DEN virus. An anamnestic response was observed in all monkeys after the second dose. No statistically significant difference in levels of neutralizing antibodies was observed between YF virus-immune and nonimmune monkeys which received the tetravalent YF/DEN1-4 vaccine or between tetravalent YF/DEN1-4-immune and nonimmune monkeys which received the YF-VAX. However, preimmune monkeys developed either no detectable viremia or a level of viremia lower than that in nonimmune controls. This is the first recombinant tetravalent dengue vaccine successfully evaluated in nonhuman primates.     

Desensitization of mouse Leydig cells in vivo: evidence for the depletion of cellular cholesterol

The study presents a characterization of the refractory state in purified mouse Leydig cells desensitized by a single injection of human chorionic gonadotropin (hCG) in vivo. The treatment of mice with 1 microgram hCG i.p. for 48 h followed by Leydig cell isolation and purification resulted in a decrease in the maxima of hCG-induced cAMP accumulation and testosterone production by approximately 70% and approximately 55%, respectively, when compared to cells of control mice. Despite a 55% reduction in 125I-hCG binding sites, the sensitivity of stimulation was not changed. The refractoriness in testosterone production in vitro was also present when the Leydig cells were stimulated with cholera toxin or dibutyryl cAMP; however, it was not observed when testosterone production was induced by the addition of pregnenolone or 20 alpha- and 22(R)-hydroxycholesterol. Mouse lipoproteins, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in natural composition, were also able to overcome the steroidogenic block (although not always completely). On the basis of the cholesterol content of the lipoproteins, the two classes were similarly effective. They increased maximal hCG-induced testosterone production not only in desensitized cells, but also in control cells (by 80-100%), whereas their effect on basal testosterone production was negligible. In desensitized cells from hCG-treated mice (2 micrograms i.p., 48 h) cellular unesterified and esterified cholesterol were decreased by 21% and 81%, respectively, when compared to control cells. This loss occurred in the face of unchanged plasma cholesterol levels. In conclusion, our data indicate that the impaired steroidogenesis in mouse Leydig cells desensitized in vivo by a single injection of hCG is the result of a depletion in cellular cholesterol, rather than of an impaired conversion of cholesterol to testosterone.     

The composition of immune sera against Neisseria meningitidis studied by high-performance liquid chromatography

The comparative study of the composition of immune rabbit sera to N. meningitidis, as well as nonimmune sera, has been made by the methods of HPLC and radial immunodiffusion. The quantitative evaluation of the main serum proteins by the two methods has shown the coincidence of the results yielded by these methods. To study the total level of IgM and IgG in the sera under study, a simple and rapid HPLC technique is proposed. The study of the stability of sera during storage (at 4-6 degrees C) has revealed that immune sera show greater stability during storage under such conditions in comparison with sera obtained from nonimmune animals.     

Natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity

Early control of virus replication by the innate immune response is essential to allow time for the generation of a more effective adaptive immune response. As an important component of innate immunity, complement has been shown to be necessary for protection against numerous microbial infections. This study was undertaken to investigate the role of complement in neutralizing influenza virus. Results demonstrated that the classical pathway of complement mediated serum neutralization of influenza virus. Although nonimmune serum neutralized influenza virus, the mechanism of virus neutralization (VN) required antibody, as sera from RAG1-deficient mice lacked VN activity; moreover, purified natural immunoglobulin M (IgM) restored VN activity to antibody-deficient sera. The mechanism of VN by natural IgM and complement was associated with virion aggregation and coating of the viral hemagglutinin receptor; however, viral lysis did not significantly contribute to VN. Additionally, reconstitution of RAG1-deficient mice with natural IgM resulted in delayed morbidity during influenza virus infection. Collectively, these results provide evidence that natural IgM and the early components of the classical pathway of complement work in concert to neutralize influenza virus and that this interaction may have a significant impact on the course of influenza viral pneumonia.     

Immunization with extracellular vesicles excreted by Toxoplasma gondii confers protection in murine infection,activating cellular and humoral responses

The study aim was to analyze whether microvesicles and exosomes, named extracellular vesicles (EVs), purified from Toxoplasma gondii are able to stimulate the protective immunity of experimental mice when administered, as challenge, a highly virulent strain. EVs excreted from T. gondii tachyzoites (RH strain) were purified by chromatography and used for immunization assays in inbred mouse groups (EV-IM). Chronic infected (CHR) and naive (NI) mice were used as control groups, since the immune response is well known. After immunizations, experimental groups were challenged with 100 tachyzoites. Next, parasitemias were determined by real-time PCR (qPCR), and survival levels were evaluated daily. The humoral response was analyzed by detection of IgM, IgG, IgG1 and IgG2a, and opsonization experiments. The cellular response was evaluated in situ by immunohistochemistry on IFN-γ, IL-10, TNF-α and IL-17 expression in cells of five organs (brain, heart, liver, spleen and skeletal muscles). EV immunization reduced parasitemia and increased the survival index in two mouse lineages (A/Sn and BALB/c) infected with a lethal T. gondii strain. EV-IM mice had higher IgG1 levels than IgM or IgG2a. IgGs purified from sera of EV-IM mice were able to opsonize tachyzoites (RH strain), and mice that received these parasites had lower parasitemias, and mortality was delayed 48 h, compared with the same results from those receiving parasites opsonized with IgG purified from NI mice. Brain and spleen cells from EV-IM mice more highly expressed IFN-γ, IL-10 and TNF-α. In conclusion, EV-immunization was capable of inducing immune protection, eliciting high production of IgG1, IFN-γ, IL-10 and TNF-α.     

Forssman antigen expressed on lymph node cells of MRL/MpJ-lpr/lpr mice is of a glycoprotein nature

This paper reports the nature of abnormally expressed Forssman (F) antigen in the lymph node cells of MRL/MpJ-lpr/lpr, autoimmune mice, and also reports its autoantibody in sera. By acetylation study of the F antigen with [14C]acetic anhydride, we concluded that the F antigen was not a glycolipid but a glycoprotein. Several bands of F-active glycoproteins were identified on a nitrocellulose sheet after purification by an anti-F antibody affinity column. Hemolysis of SRBC by some sera from MRL/MpJ/lpr/lpr was inhibited by purified F glycoprotein and also by F glycolipid. The antibody in the serum, however, seemed to be more specific for F glycoproteins than F glycolipid, but the opposite was the case for rabbit anti-F glycolipid antibody. No significant difference of the SRBC hemolysis levels was observed between the sera from MRL/MpJ-lpr/lpr and its congenic MRL/MpJ-+/+ mice.     

Unimpaired coupling of phosphorylated, desensitized beta-adrenoceptor to Gs in a reconstitution system

Heterologous desensitization of turkey erythrocyte beta-adrenoceptors correlates with receptor phosphorylation and impaired receptor-Gs coupling, as assessed by fusion of purified desensitized receptors with X. laevis erythrocytes [(1984) Science 225, 837-840]. We have purified beta-receptors from desensitized and untreated turkey erythrocytes and have compared the abilities of these two receptors to couple with pure turkey erythrocyte Gs in a reconstituted system. Functional receptor-Gs coupling was assessed by measuring hormone-dependent Gs activation by GTP gamma S and GTPase activity. While in membranes prepared from desensitized cells, receptor-Gs coupling was clearly reduced, this effect was absent when coupling of purified desensitized receptor was measured. We conclude that covalent modification by phosphorylation does not fully explain the functional uncoupling at the membrane level.