The induction of at least two distinct types of interferon in mouse spleen cell cultures by Corynebacterium parvum

Mice immunized with particulate antigens or soluble antigens in Freund's complete adjuvant produce a factor(s) which enhances antibody formation. Such an enhancing factor(s) is detected in the serum within 6 hr after immunization. The factor(s) is specific and enhances both 19s and 7s responses especially when recipient mice are challenged with subimmunogenic doses of antigen. From the study of the kinetics of antibody formation (latent period, coincidence of peaks of 19s and 7s responses) and the distribution of the Ig classes of the antibody (dominance of IgG1) it is concluded that the enhancing factor(s) primes the animals for a secondary response. The enhancing factor(s) is carrier specific and enhances antibody formation in the absence of T cells (nude mice). In the absence of T cells the antibody response is quantitatively small, which suggests that in the presence of T cells the enhancing factor(s) further amplifies antibody formation.     

Acellular pertussis booster in adolescents induces Th1 and memory CD8+ T cell immune response

In a number of countries, whole cell pertussis vaccines (wcP) were replaced by acellular vaccines (aP) due to an improved reactogenicity profile. Pertussis immunization leads to specific antibody production with the help of CD4(+) T cells. In earlier studies in infants and young children, wcP vaccines selectively induced a Th1 dominated immune response, whereas aP vaccines led to a Th2 biased response. To obtain data on Th1 or Th2 dominance of the immune response in adolescents receiving an aP booster immunization after a wcP or aP primary immunization, we analyzed the concentration of Th1 (IL-2, TNF-α, INF-γ) and Th2 (IL-4, IL-5, IL-10) cytokines in supernatants of lymphocyte cultures specifically stimulated with pertussis antigens. We also investigated the presence of cytotoxic T cell responses against the facultative intracellular bacterium Bordetella pertussis by quantifying pertussis-specific CD8(+) T cell activation following the aP booster immunization. Here we show that the adolescent aP booster vaccination predominantly leads to a Th1 immune response based on IFNgamma secretion upon stimulation with pertussis antigen, irrespective of a prior whole cell or acellular primary vaccination. The vaccination also induces an increase in peripheral CD8(+)CD69(+) activated pertussis-specific memory T cells four weeks after vaccination. The Th1 bias of this immune response could play a role for the decreased local reactogenicity of this adolescent aP booster immunization when compared to the preceding childhood acellular pertussis booster. Pertussis-specific CD8(+) memory T cells may contribute to protection against clinical pertussis.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

The production of anti-hapten antibody after immunization with trinitrophenylated (TNP) hamster erythrocytes (HRBC) or sheep erythrocytes (SRBC) was determined in high- and low-responder mouse strains against HRBC antigen. 1) Anti-TNP antibody was detected in sera of high-responder DDD and CF1 mice after primary immunization with TNP-HRBC, but not in those of low-responder C57BL/6 mice. 2) Anti-TNP antibody was detectable in sera of all the strains after primary immunization with TNP-SRBC. 3) Production of anti-TNP antibody was elicited after a booster injection of TNP-HRBC in low-responder C57BL/6 mice pre-sensitized with HRBC in Freund's complete adjuvant. These results suggest that functions of thymus-derived cells specific for HRBC antigen are deficient in low-responder mice.     

Antibody formation in mouse bone marrow. IX. Peripheral lymphoid organs are involved in the initiation of bone marrow antibody formation.

Mouse bone marrow is barely capable of plaque-forming cell (PFC) activity during the primary response to sheep red blood cells (SRBC). However, during secondary-type responses, it becomes the major organ, containing IgM, IgG, and IgA PFC. In the present paper, the influence of splenectomy (Sx) upon the secondary bone marrow PFC response to SRBC was investigated. When previously primed mice were splenectomized just before the second intravenous (iv) injection of SRBC, the effect of Sx upon the height of the bone marrow PFC response was dependent on the booster dose. Sx just before a booster of 10⁶ SRBC iv almost completely prevented bone marrow PFC activity, whereas an iv booster dose of 4 × 10⁸ SRBC evoked a normal IgM, IgG, and IgA PFC response in Sx mice. Apparently low doses of iv administered antigen require the spleen in order to evoke antibody formation in the bone marrow. Experiments with parabiotic mice, consisting of Sx and sham-Sx mice, showed that this facilitating influence of the spleen upon bone marrow antibody formation occurs via the blood stream. In a subsequent study, it was investigated whether the spleen is required throughout the bone marrow PFC response or only during the few days of the initiation phase. Therefore, mice were splenectomized at different intervals after a booster injection of 10⁶ SRBC iv. It appeared that Sx 2 days after the booster injection could still prevent the normal bone marrow PFC activity, whereas Sx at Day 4 could no longer do so. Apparently, after an iv booster injection, the spleen is only required for initiation of the bone marrow PFC response and not for the maintenance of this PFC activity thereafter.     

Intestinal antibody to Vibrio cholerae in immunised mice.

The immune response of the mouse to priming and booster doses of V. cholerae was studied to establish whether serum antibody could be used as a correlate of local immunity. Serum antibody titres following oral boosting of orally-primed animals were shown to reflect the state of local intestinal immunity. This was not the case when the same oral booster dose was given to parenterally-primed animals. These results were discussed in relation to the human endemic situation. The highest titres of intestinal protective antibodies were found following combination of the oral and parenteral routes of immunisation. Various killed or extracted preparations of V. cholerae were used as oral vaccines to test their ability to induce protective antibodies in the gut. Only Boivin antigen was capable of inducing as good an intestinal antibody response as would the living organism.     

Targeting and cellular trafficking of magnetic nanoparticles for prostate cancer imaging

Antibody-conjugated iron oxide nanoparticles offer a specific and sensitive tool to enhance magnetic resonance (MR) images of both local and metastatic cancer. Prostate-specific membrane antigen (PSMA) is predominantly expressed on the neovasculature of solid tumors and on the surface of prostate cells, with enhanced expression following androgen deprivation therapy. Biotinylated anti-PSMA antibody was conjugated to streptavidin-labeled iron oxide nanoparticles and used in MR imaging and confocal laser scanning microscopic imaging studies using LNCaP prostate cancer cells. Labeled iron oxide nanoparticles are internalized by receptor-mediated endocytosis, which involves the formation of clathrin-coated vesicles. Endocytosed particles are not targeted to the Golgi apparatus for recycling but instead accumulate within lysosomes. In T(1)-weighted MR images, the signal enhancement owing to the magnetic particles was greater for cells with magnetic particles bound to the cell surface than for cells that internalized the particles. However, the location of the particles (surface vs internal) did not significantly alter their effect on T(2)-weighted images. Our findings indicate that targeting prostate cancer cells using PSMA offers a specific and sensitive technique for enhancing MR images.     

IgE and IgG are synergistic in antigen-mediated release of thromboxane from human lung macrophages.

The mechanism of IgE-mediated release of thromboxane A2 from human lung macrophages has been studied using a monoclonal chimeric human/mouse IgE antibody and its specific antigen. The cells could be sensitized at 37 degrees C but not at 4 degrees C by incubation with IgE, and released a significant amount of thromboxane A2 (TXA2), measured as the stable hydrolysis product TXB2, in response to an anti-chimeric IgE antibody. In contrast, stimulation of IgE-sensitized macrophages with the specific antigen produced less than 10% of this response. A similar time course for the release of TXB2 and the formation of inositol monophosphate in the presence of LiCl was observed. Cleavage of the Fc domain of the anti-chimeric IgE antibody substantially eliminated its capacity to stimulate IgE-sensitized cells. However, the weak or undetectable response to chimeric IgE plus specific antigen was substantially potentiated by an antigen-specific chimeric IgG antibody. IgG-sensitized macrophages did not respond to antigen challenge by the release of TXB2. Preincubation of the cells with a monoclonal antibody against the low affinity receptor for IgE (Fc epsilon RII/CD23) did not prevent IgE sensitization. We conclude that cell-bound IgE antibody cannot induce the release of TXB2 but has fixed antigen which then must interact with specific IgG antibody and IgG receptors to induce mediator release.     

Conditions of development of a secondary immune response in guinea pig lymphoid cell cultures]

On addition of a specific antigen to the cell culture of the lymph nodes of the immunized guinea pigs there was seen an intensification of the synthesis of the DNA and of the antibody formation. Both processes occurred more intensively in incubation of cells on a medium with an addition of rabbit antiserum to the guinea pig erythrocytes than on a medium with a normal homologous serum. It is supposed that the optimal conditions for the initiation of antibody formation in the lymphoid cells were created in case of a combined action of a specific antigen-inductor and a nonspecific stimulant intensifying the DNA synthesis.     

A novel antigen is shared by retinal pigment epithelium and pigmented neural crest

 Pigment cells in vertebrate embryos are formed in both the central and peripheral nervous system. The neural crest, a largely pluripotent population of precursor cells derived from the embryonic neural tube, gives rise to pigment cells which migrate widely in head and trunk.The retinal pigment epithelium is derived from the optic cup, which arises from ectoderm of the neural tube. We have generated an antibody, ips6, which stains an antigen common to pigment cells of retinal pigment epithelium and neural crest. Ips6 stains retinal pigment epithelium and choroid as well as a subset of crest cells that migrate in pathways typical of melanoblasts. Immunoreactivity is seen first in the eye and later in a subset of migrating crest cells. Crest cells in the amphibian embryo migrate along specific, stereotyped routes; ips6 immunoreactive cells are found in some but not all of these pathways. In older wild-type embryos, cells expressing ips6 appear coincident with pigment-containing cells in the flank, head, eye and embryonic gut. In older animals, staining in the eye extends to the intraretinal segment of optic nerve and interstices between photoreceptors and cells at the retinal periphery. We suggest that the ips6 antibody defines an antigen common to pigment cells of central and peripheral origin. Received: 22 January 1996/Accepted: 15 July 1996     

Transfer of memory cells into antigen-pretreated hosts. I. Functional detection of migration sites for antigen-specific B cells.

The distribution of functionally active hapten-specific B memory cells was investigated. Using antigen-pretreated lethally irradiated recipients, a marked accumulation of adoptively transferred B memory cells was demonstrated in lymph nodes containing specific antigen, but not in lymph nodes containing non-cross-reacting hapten conjugates. This difference in responsiveness between lymph nodes containing specific versus those containing nonspecific antigen developed over a period 3–5 days after memory cell transfer. The localization of antigen specific cells was T-cell independent; both carrier-primed T helper cells and specific antigenic challenge, however, were required to trigger the localized B memory cells into antibody production. Specific B memory cell accumulation did not result from an expansion of the antigen-specific cell population due to local proliferation induced by antigen depots in the lymph nodes to challenge. Rather, the results indicated that recirculating B memory cells had progressively accumulated through retention by antigen in the lymph node. These findings suggest that, in the absence of T-cell help and specific antigenic challenge, B memory cells accumulate in lymphoid tissue (follicles) without responding and provide persistent local memory for the humoral immune response.     

Human peripheral blood lymphocytes from recently vaccinated individuals produce both type-specific and intertypic cross-reacting neutralizing antibody on in vitro stimulation with one type of poliovirus

An in vitro system of poliovirus-specific antibody production by peripheral blood B cells on stimulation by the virus has been developed. Virus-neutralizing antibodies in culture supernatant fluids, or virus-specific antibody-secreting cells (ASC) were detected by microneutralization assay and ELISA-SPOT test, respectively. After booster immunization with polio vaccine, anti-poliovirus-neutralizing ASC were present in circulation. This response was measurable between 5 and 12 days after booster vaccination. At between 12 and 90 days, another subset of B cells was found in peripheral blood that only produced poliovirus-specific neutralizing antibody after in vitro antigenic stimulation. The in vitro virus-induced response required B cells, monocytes, and T4+ (T helper) cells, and was shown to result from de novo protein synthesis. The anti-poliovirus-neutralizing response in vitro could be dissected in a type-specific and intertypic cross-reactive response by using various antigen concentrations for in vitro stimulation. Evidence was obtained by absorption studies for the existence of intertypic cross-reactive neutralization-inducing epitopes.     

Susceptibility of astrocytes to class I MHC antigen-specific cytotoxicity

Cell-mediated immune mechanisms contribute to tissue injury within the central nervous system (CNS) in a number of experimental diseases, including experimental allergic encephalomyelitis and some viral infections, and may mediate lesion formation in multiple sclerosis. We investigated the conditions under which murine astrocytes can become susceptible targets of cytotoxic T cells. We demonstrate that mouse astrocytes in vitro can be susceptible targets of class I major histocompatibility complex (MHC)-specific cytotoxicity mediated by L3 cytotoxic T lymphocytes (CTL). Expression of appropriate class I MHC antigen on the astrocytes is a requirement, because only cells bearing the H-2d phenotype are susceptible to lysis by L3 cells. BALB/c-H-2dm2 astrocytes lacking the specific determinant recognized by L3 cells are not susceptible to lysis. Astrocyte lysis can, however, occur under culture conditions in which MHC antigen expression is immunocytochemically low or undetectable. Cytolysis can be inhibited by pretreatment of the effector L3 cells with either anti-Lyt-2 monoclonal antibody (mAb) or anti-clonotypic mAb and by preincubation of the glial target cells with an appropriate anti-H-2 antibody (anti-H-2Ld). mAb to lymphocyte function-associated antigen does not inhibit cytotoxicity of the L3 clone against glial cells. Knowledge regarding the role of CTL within the CNS, including the surface molecules involved in glial cell lysis, could further the development of immunotherapies designed to effect immune reactivity within the CNS.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

Golden hamsters were used as hosts in this work, and mice of various strains as donors of antigens. 1) There were no strain differences in immunogenicity of erythrocytes from C57BL/6, AKR, SL and CF1 mice. 2) Primary intravenous immunization with mouse erythrocytes (MRBC) induced the production of hemolysin plaque-forming cells (PFC) in a large number, but elicited only in a negligible titer production of 2-mercaptoethanol-resistant antibody. 3) 2-Mercaptoethanol-resistant antibody was produced more efficiently in hamsters pre-sensitized with mouse lymph node (MLN) cells rather than those pre-immunized with MRBC after a booster with MRBC. 4) Numbers of PFC in pre-sensitized hamsters were three-times that of the non-sensitized hamsters after a booster with MRBC, when pre-sensitization was performed intradermally with a small number of MLN cells. 5) Average diameter of the hemolysin plaques in pre-sensitized hamsters was one and a half times larger than that in non-sensitized hamsters. Conclusions agree well with the results in our previous papers that the reversed combination of hosts and antigen donors employed support the concept that certain processes required for delayed hypersensitivity contributed to antibody production under a condition suitable for antibody response.     

Antibody response of dogs inoculated with a large number of cultured canine monocytes adsorbed with a calicivirus in advance

Five conventional Beagle dogs were intravenously injected with ten million canine monocyte cells (Cn/K99) cultured in vitro (Kadoi, 2000) adsorbed with a strain of calicivirus originally isolated from lions (Kadoi et al., 1997). Another two Beagle dogs were injected similarly with the virus suspension solely as control. Serum samples were collected from these dogs at intervals and specific seroneutralizing antibody production against the virus was measured in vitro. A significantly higher antibody production was demonstrated in the five dogs group. A clear booster effect was also proved in the sera of the dogs after the second virus inoculation made on day 100. A possibility of antigen presentation function of non-self monocytes is suggested.     

Effect of Statolon-induced Interferon on Antibody Formation

The effect of statolon-induced interferon on immune response was investigated. Young adult Swiss albino mice were immunized with chicken red blood cells or infectious mengovirus. The red blood cells were injected three times and the virus twice at 2-week intervals, with or without treatment with statolon 24 hr before the inoculations. The effect of interferon on antibody formation was measured by determinations of hemagglutinating, hemolytic, and virus-neutralizing antibodies in the sera of treated and nontreated groups. The hemagglutinin and hemolysin titers after each successive inoculation were equal in both groups, indicating that interferon had no effect on their production. A decrease in the primary response to the viral antigen was associated with interferon induction; the booster response was unaffected. The possible use of interferon inducers as attenuants for immunization with live virulent viruses is discussed.     

The induction and suppression of in vitro IgG anti-tetanus toxoid antibody synthesis by human lymphocytes stimulated with tetanus toxoid in the absence of in vivo booster immunizations

This report presents a new approach that by-passes booster immunizations with tetanus toxoid (TT) before in vitro studies of antibody (Ab) production. The methodology for optimal TT-induced synthesis of specific IgG anti-tetanus toxoid Ab (IgG anti-TT) by peripheral blood mononuclear cells (PBMC) from randomly selected TT immune individuals without recent booster immunizations is described. PBMC from most normal immune subjects could be repeatedly induced to produce in vitro IgG anti-TT; PBMC from subjects with high TT titers are not required for this new approach. This approach uses high cell concentrations in multiple replicate microcultures and TT washout to obtain optimal IgG anti-TT synthesis. Washed cultures produced more Ab than nonwashed cultures (p less than or equal to 0.005). The readdition of TT (2.5 to 250 ng/ml) to the culture media after washout of TT on day 4 suppressed specific Ab formation, whereas diphtheria toxoid added at comparable doses did not inhibit specific Ab formation. Suppression of antibody synthesis mediated by T cells could be induced by TT per se, and was not due to binding of synthesized Ab to TT in the latter 8 days of culture. In addition, suppression could not be induced in the first 4 days of culture by IgG anti-TT, IgG, or IgM. This approach permits the analysis of antigen-specific regulatory circuits in the steady and activated immune states, and the evaluation of in vivo and in vitro effects of biologic response modifiers on specific Ab production.     

Trichinella spiralis: secreted antigen of the infective L1 larva localizes to the cytoplasm and nucleoplasm of infected host cells

Antibodies were elicited against a purified antigen with an apparent molecular weight of 43K. This antibody preparation also detected a second antigen consisting of a group of closely related components of 45-50K. These antigens are stage specific for the infective first stage larva of Trichinella spiralis and are among the repertoire of secreted antigens originating from the stichosome. Antibody raised against the 43K antigen reacted with the stichosome and cuticle of the mature larva and the cytoplasm and nucleoplasm, but not nucleolus, of all nuclei of infected host cells (Nurse cells) in sections of infected tissues. Studies on sections of synchronously infected muscle tissue revealed that antigen was present only within the worm on Day 7 of the infection. On Day 9 after infection, the stichosome and cuticular surface of the larva and the cytoplasm and nucleoplasm of each nucleus of the Nurse cell reacted with antibody. Nurse cell cytoplasmic and nuclear reactivity increased in intensity until Day 18 after infection. These results suggest that stichocyte-specific antigens are synthesized during the early phase of infection in the muscle, and that as the Nurse-parasite complex develops, some of the antigen is secreted into the milieu of the Nurse cell. The presence of antigen in the cytoplasm and nucleoplasm of the infected host cell is discussed in relation to Nurse cell formation and maintenance.     

Human growth hormone presented by K14hGH-transgenic skin grafts induces a strong immune response but no graft rejection

Although immune responses leading to rejection of transplantable tumours have been well studied, requirements for epithelial tumour rejection are unclear. Here, we use human growth hormone (hGH) expressed in epithelial cells (skin keratinocytes) as a model neo-self antigen to investigate the consequences of antigen presentation from epithelial cells. Mice transgenic for hGH driven from the keratin 14 promoter express hGH in skin keratinocytes. This hGH-transgenic skin is not rejected by syngeneic non-transgenic recipients, although an antibody response to hGH develops in grafted animals. Systemic immunization of graft recipients with hGH peptides, or local administration of stimulatory anti-CD40 antibody, induces temporary macroscopic graft inflammation, and an obvious dermal infiltrate of inflammatory cells, but not graft rejection. These results suggest that a neo-self antigen expressed in somatic cells in skin can induce an immune response that can be enhanced further by induction of specific immunity systemically or non-specific immunity locally. However, immune responses do not always lead to rejection, despite induction of local inflammatory changes. Therefore, in vitro immune responses and in vivo delayed type hypersensitivity are not surrogate markers for immune responses effective against epithelial cells expressing neoantigens.     

The Rosy Locus in Drosophila Melanogaster: Xanthine Dehydrogenase and Eye Pigments

The rosy gene in Drosophila melanogaster codes for the enzyme xanthine dehydrogenase (XDH). Mutants that have no enzyme activity are characterized by a brownish eye color phenotype reflecting a deficiency in the red eye pigment. Xanthine dehydrogenase is not synthesized in the eye, but rather is transported there. The present report describes the ultrastructural localization of XDH in the Drosophila eye. Three lines of evidence are presented demonstrating that XDH is sequestered within specific vacuoles, the type II pigment granules. Histochemical and antibody staining of frozen sections, as well as thin layer chromatography studies of several adult genotypes serve to examine some of the factors and genic interactions that may be involved in transport of XDH, and in eye pigment formation. While a specific function for XDH in the synthesis of the red, pteridine eye pigments remains unknown, these studies present evidence that: (1) the incorporation of XDH into the pigment granules requires specific interaction between a normal XDH molecule and one or more transport proteins; (2) the structural integrity of the pigment granule itself is dependent upon the presence of a normal balance of eye pigments, a notion advanced earlier.     

Antibody formation in experimental kidney failure and in the early stages of restorative kidney growth in mice

The ability of spleen cells to respond with antibody formation to a foreign antigen (sheep erythrocytes) was studied in mice at different kidney lesions (uni- and bilateral nephrectomy, ureter ligation, pseudo-operation, wound of one kidney) during the early postoperation period (1-72 h). In the case of bilateral nephrectomy, the reliable increase in the number of antibody-forming cells (AFC) was noted already within 1 h after the operation, in the case of unilateral nephrectomy within 12 h. In the case of bi- and unilateral ligations of ureter, the response was delayed by 3 and 5 h, respectively. Sham operation and kidney wound did not stimulate antibody formation. It is suggested that the antibody-forming ability of the spleen cells does not depend on stress, renal deficiency or destructive changes and that the antibody formation is activated by disturbances in the ratio of immunoregulatory cells.