In vitro parasite antigen-induced antibody responses in human helminth infections

Helminth parasites characteristically induce vigorous antibody responses in human infections, but the immunoregulatory mechanisms determining the level of these responses are not at all understood. To investigate these mechanisms, peripheral blood mononuclear cells were obtained from 10 patients with parasitic helminth infections (three with schistosomiasis, three with onchocerciasis, and four with loiasis), along with six normal controls. These cells were then cultured in vitro and the capacity of the cells to respond to a specific parasite antigenic stimulus was examined by measuring the amount of parasite-specific antibody produced. Parasite antigen alone, without exogenous mitogen, induced an IgG anti-parasite antibody response in vitro. Optimal responses were obtained at extremely low antigen concentrations--concentrations at which little if any polyclonal immunoglobulin production occurred. Additionally, the in vitro induction of parasite-specific antibody was antigen dose-dependent, requiring much lower antigen concentrations than those necessary to induce lymphocyte blastogenesis. Antibody production was shown to require the cooperative interaction of B and T cells. These studies demonstrate that in vitro responses to antigens from naturally acquired parasitic infections, like those in individuals postimmunization, can be utilized to dissect the cellular and humoral factors that regulate antibody production to naturally acquired human pathogens.     

Glycan microarray profiling of parasite infection sera identifies the LDNF glycan as a potential antigen for serodiagnosis of trichinellosis

Diagnostic methods for parasite infections still highly depend on the identification of the parasites by direct methods such as microscopic examination of blood, stool and tissue biopsies. Serodiagnosis is often carried out to complement the direct methods; however, few synthetic antigens with sufficient sensitivity and specificity are available. Here we evaluated a glycan microarray approach to select for synthetic glycan antigens that could be used for serodiagnosis of parasitic infections. Using a glycan array containing over 250 different glycan antigens, we identified GalNAcβ1–4(Fucα1–3)GlcNAc-R (LDNF) as a glycan antigen that is recognized by antibodies from Trichinella-infected individuals. We synthesized a neoglycoconjugate, consisting of five LDNF molecules covalently coupled to bovine serum albumin (BSA), and used this neoglycoconjugate as an antigen to develop a highly sensitive total-Ig ELISA for serological screening of trichinellosis. The results indicate that glycan microarrays constitute a promising technology for fast and specific identification of parasite glycan antigens to improve serodiagnosis of different parasitic infections, either using an ELISA format, or parasite-specific glycan arrays.     

Lymphocyte Stimulation and Leucocyte Migration Inhibition as Diagnostic Tools for the Detection of Mycobacterium Avium Infection in Cattle

The tuberculin skin test is the conventional method of detecting infections with mycobacteria in animals. A positive reaction is considered to reflect cell-mediated immunity (CMI). CMI against mycobacteria can be studied by in vitro systems using suspensions of blood lymphocytes or leucocytes. The reactivity of these cells to different antigens can be measured in the lymphocyte stimulation (LS) (Muscoplat et al 1975, Bergman 1976, Johnson & Morein 1976), or leucocyte migration inhibition (LMI) (Aalund 1970, Clausen 1973) tests.     

Influence of maternal infection on offspring resistance towards parasites

Immunoglobulins, parasite circulating antigens, immune cells, cytokines and other cell-related products can be transferred from infected mothers to their young. They can combine their effects to interact with the invading parasites, as well as to induce a long-term modulation of the offspring's capacity to mount an immune response to subsequent exposure to parasites. The protective effect of maternally derived antibodies may be limited by the selective transfer of immunoglobulin isotypes. Maternal antibodies may also prevent the priming of specific cells in offspring or inhibit the progeny's antibody production by interacting with B-cell receptors or with the idiotypic repertoire. The potentially beneficial priming effect of transferred parasitic antigens may be altered by the Th2-cell-biased foetal environment and such antigens may also induce deletion or anergy of T- and B-cell clones in offspring. Therefore, besides protective effects, maternal infection may downregulate the offspring's immune response. If such hyporesponsiveness may be clearly harmful (in increasing the risk or in worsening congenital or postnatally acquired infections in offspring), it can also be beneficial (in limiting the pathogenesis of some infections). Here, Yves Carlier and Carine Truyens review the rationale of these complex foeto-maternal relationships in parasitic diseases.     

The regulation of immunological responses to parasitic infections and the development of tolerance.

It is well established that specific unresponsiveness to immunization can be induced by prolonged exposure to antigenic proteins. More generally, many parasitic infections, such as the helminth worms and the Leishmania parasites, appear to be able to persist in some of their human hosts over long periods of time, via what appears to be an ability to induce defective or inappropriate T-cell responses (= tolerance). Recent research has suggested that cytokines, produced by specific subsets of CD4+ T-cells (characterized by cytokine secretory profiles and growth properties), have an important, and often complex, role in promoting or inhibiting host protective immunity to parasitic infections. By examination of the population dynamics of the stimulation and regulation of cellular responses to infection, via the use of simple mathematical models, we show that nonlinear interactions between CD4+ T-cell subsets and their secreted cytokines can result in either host protection or immunological unresponsiveness, depending on the magnitude and duration of exposure to parasitic infection. Analyses also identify a possible mechanism to explain the stimulation of two separate peaks of enhanced T-cell-mediated responses over a wide range of levels of antigenic exposure.     

Time-dependent mode of immunization augments suppressed antibody responses in spleen cell cultures from mice experimentally infected with Trypanosoma cruzi

Mice infected with Trypanosoma cruzi develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC). After normal or infected mice were primed with SRBC, their spleen cells were restimulated 4 days later with SRBC in Mishell-Dutton cultures and found to mount hyperaugmented IgM anti-SRBC responses. It was also demonstrated that T-cells derived from normal mice primed in vivo 4 days previously with SRBC, and subsequently added to cultures of spleen cells from T. cruzi-infected mice, enhanced anti-SRBC DPFC responses in a dose-dependent fashion. These results show that functional help provided by T-cells activated during an in vivo priming and exposed to an in vitro challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in the spleen cell cultures from T. cruzi-infected mice.     

Trypanosoma cruzi: maintenance of parasite-specific T cell responses in lymph nodes during the acute phase of the infection

Mice infected with 5 x 10(3) forms of Trypanosoma cruzi showed a transient, but severe impairment of in vitro spleen cell responses to parasite antigens and to Concanavalin A (Con A). In contrast, inguinal and periaortic lymph node (LN) cells displayed high parasite-specific proliferative responses and only a partial reduction of the Con A-induced proliferation during the acute and chronic phases of infection. Lymphocytes that underwent blastic transformation in T. cruzi-stimulated cell cultures were of the L3T4+ phenotype. Suppression of spleen cell responses occurred in the acute phase whether mice were infected with high (3 x 10(5] or low (5 x 10(3] doses of T. cruzi by intraperitoneal or subcutaneous route. Suppression of the T. cruzi-specific proliferative response of LN cells was only observed in mice infected with high subcutaneous inocula. This suppression, however, was restricted to the LNs draining the site of inoculation without affecting distant LNs. Supernatants from parasite-stimulated proliferating LN cells displayed low or undetectable T cell growth factor (TCGF) activity, in contrast with the high TCGF levels found in supernatants of the same cells stimulated with Con A. Low levels of TCGF were also detected in cultures of LN cells from mice immunized with T. cruzi extracts. Neither the T. cruzi antigen used for in vitro stimulation nor the LN cell supernatants from infected mice inhibited TCGF activity. These findings indicate that (1) parasite-specific responses are present in the LN compartment throughout the acute phase of T. cruzi infection in mice and (2) the proliferative response of L3T4+ LN cells from infected mice to T. cruzi antigens is not associated with a high TCGF secretory response.     

Lymphokines in sensitized rats. III. Influence of prednisolone treatment of lymph node and spleen architecture in sensitized rats and upon MIF release in vitro.

The influence of prednisolone (corticosteroid, C.S.) treatment upon established cell-mediated immunity (CMI) induced by complete Freund's adjuvant (CFA) has been studied in rats by using in vitro migration inhibitory factor (MIF) assays, type IV skin reactions, and regional lymph node and spleen histology. Additionally, changes in the mononuclear-polymorphonuclear ratio of peripheral blood and T-cell accumulation in bone marrow in response to C.S. treatment have been determined. These results have been evaluated by comparison with equivalent experiments upon animals treated with anti-rat lymphocyte serum (ALS), oxisuran, or 2-[(methylsulfinyl)-acetyl]pyridine, which selectively suppresses CMI. The results suggest the existence of a population of “educated” T-cells in the thymic cortex of sensitized rats, and they suggest that prolonged C.S. administration does not suppress T-effector cells involved in established CMI but, rather, affects lymphocyte and monocyte migration patterns, including the migration of educated T-cells from the thymic cortex into other tissue compartments.     

Time-dependent mode of immunization augments suppressed antibody responses in spleen cell cultures from mice experimentally infected with Trypanosoma cruzi

Mice infected with Trypanosoma cruzi develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC). After normal or infected mice were primed with SRBC, their spleen cells were restimulated 4 days later with SRBC in Mishell-Dutton cultures and found to mount hyperaugmented IgM anti-SRBC responses. It was also demonstrated that T-cells derived from normal mice primed in vivo 4 days previously with SRBC, and subsequently added to cultures of spleen cells from T. cruzi-infected mice, enhanced anti-SRBC DPFC responses in a dose-dependent fashion. These results show that functional help provided by T-cells activated during an in vivo priming and exposed to an in vitro challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in the spleen cell cultures from T. cruzi-infected mice.     

Do Antenatal Parasite Infections Devalue Childhood Vaccination?

On a global basis, both potent vaccine efficacy and high vaccine coverage are necessary to control and eliminate vaccine-preventable diseases. Emerging evidence from animal and human studies suggest that neglected tropical diseases (NTDs) significantly impair response to standard childhood immunizations. A review of efficacy and effectiveness studies of vaccination among individuals with chronic parasitic infections was conducted, using PUBMED database searches and analysis of data from the authors'' published and unpublished studies. Both animal models and human studies suggest that chronic trematode, nematode, and protozoan infections can result in decreased vaccine efficacy. Among pregnant women, who in developing countries are often infected with multiple parasites, soluble parasite antigens have been shown to cross the placenta and prime or tolerize fetal immune responses. As a result, antenatal infections can have a significant impact on later vaccine responses. Acquired childhood parasitic infections, most commonly malaria, can also affect subsequent immune response to vaccination. Additional data suggest that antiparasite therapy can improve the effectiveness of several human vaccines. Emerging evidence demonstrates that both antenatal and childhood parasitic infections alter levels of protective immune response to routine vaccinations. Successful antiparasite treatment may prevent immunomodulation caused by parasitic antigens during pregnancy and early childhood and may improve vaccine efficacy. Future research should highlight the varied effects that different parasites (alone and in combination) can have on human vaccine-related immunity. To optimize vaccine effectiveness in developing countries, better control of chronic NTDs may prove imperative.     

Immunocytochemical characterization of S-100 beta-positive human T-lymphocytes by a double immunostaining method

The antigenic properties of S-100 beta-positive human T-lymphocytes (S-100 beta+ T-cells) were investigated by a double immunostaining technique employing an indirect immunoperoxidase method for cytoplasmic S-100 beta subunit and an immunoalkaline phosphatase method for cell surface antigens detected by various monoclonal antibodies to human lymphocytes. S-100 beta+ T-cells recognized by their diffuse intracytoplasmic immunoperoxidase reaction, also expressed CD2, CD3, CD8 antigens demonstrated by surface blue alkaline phosphatase reactivity, but not CD4, CD1, CD25 (interleukin-2 receptor), or HLA-DR antigens. However, they displayed a blastic change to T-cell mitogens, such as Concanavalin A(Con-A) and PHA, followed by the expression of CD25 and HLA-DR antigens. Under normal conditions, S-100 beta+ T-cells comprised approximately 5-22.8% of CD8+ cells amongst human peripheral blood mononuclear cells.     

Heterogeneity in expression of human leukocyte antigens and melanoma-associated antigens in advanced melanoma

The study of tumor immunology has led to many innovative therapeutic strategies for the treatment of melanoma. The strategies are primarily dependent on melanoma-associated antigen peptide vaccination or T-cell-based therapy. These immunotherapies are totally reliant on proper copresentation of human leukocyte antigen class I molecules in sufficient quantity and the presence and availability of melanoma-associated antigenic peptides. Altered expression of either HLA class I molecules or melanoma antigens is known to occur. These defects lead to altered manufacture and copresentation of HLA class I molecules with melanoma-associated antigens to T-cells. Defects in any one combination can lead to loss of recognition of melanoma cells and their subsequent destruction by cytotoxic T-lymphocytes. Thus, these immunotherapy strategies can be thwarted by defects or heterogeneity of expression of human leukocyte antigen class I or of melanoma-associated antigens.     

Genetic mapping reveals Nfkbid as a central regulator of humoral immunity to Toxoplasma gondii

Protective immunity to parasitic infections has been difficult to elicit by vaccines. Among parasites that evade vaccine-induced immunity is Toxoplasma gondii, which causes lethal secondary infections in chronically infected mice. Here we report that unlike susceptible C57BL/6J mice, A/J mice were highly resistant to secondary infection. To identify correlates of immunity, we utilized forward genetics to identify Nfkbid, a nuclear regulator of NF-κB that is required for B cell activation and B-1 cell development. Nfkbid-null mice (“bumble”) did not generate parasite-specific IgM and lacked robust parasite-specific IgG, which correlated with defects in B-2 cell maturation and class-switch recombination. Though high-affinity antibodies were B-2 derived, transfer of B-1 cells partially rescued the immunity defects observed in bumble mice and were required for 100% vaccine efficacy in bone marrow chimeric mice. Immunity in resistant mice correlated with robust isotype class-switching in both B cell lineages, which can be fine-tuned by Nfkbid gene expression. We propose a model whereby humoral immunity to T. gondii is regulated by Nfkbid and requires B-1 and B-2 cells for full protection.     

The specificity of nematode allergens in the diagnosis of human visceral larva migrans.

Extracts of nematodes have been used as skin test antigens in the diagnosis of nematode infections for many years. Ascaris lumbricoides and Toxocara canis are two nematodes commonly involved in human parasitism, the latter being associated with the clinical condition of Visceral Larva Migrans. In vitro and in vivo experiments reported in this paper confirm experimentally, as well as clinically, the existence of cross-reacting antigens between T. canis and Ascaris spp., and probably between T. canis and other nematodes. These cross-reactions compromise the usefulness of skin tests in the diagnosis of such parasitic infections.     

Assessment of the status of cell-mediated immunity in cytomegalovirus-infected renal allograft recipients.

Renal allograft recipients are unusually susceptible to cytomegalovirus (CMV) infections. Since humoral immunity to CMV is uncompromised in these patients, it was felt desirable to assess the competence of cell mediated immunity (CMI). Several parameters were used. On skin testing with candida, SK-SD, mumps, and PPD-5 antigens, 80.0% of patients and 5.0% of controls were unreactive. T-lymphocyte ratios (SRBC rosette test) were 18.7% in transplant patients, vs. 40.3% in controls. These differences are statistically significant. Lymphocyte stimulation assay ([³H] thymidine uptake) was developed to study CMI to CMV. Lymphocytes from all the normal seropositive subjects (10) had increased [³H]thymidine uptake on exposure to CMV antigens. There was no antigen specific stimulation of lymphocytes from the seronegative controls (five). Six of nine (67.7%) CMV infected renal allograft recipients, studied six or more months post-transplantation, had no evidence of CMI to CMV by this assay.     

The role of eosinophils in parasitic helminth infections: insights from genetically modified mice

Eosinophilia - an increase in the number of eosinophils in the blood or tissues - has historically been recognized as a distinctive feature of helminth infections in mammals. Yet the precise functions of these cells are still poorly understood. Many scientists consider that their primary function is protection against parasites, although there is little unequivocal in vivo evidence to prove this. Eosinophils are also responsible for considerable pathology in mammals because they are inevitably present in large numbers in inflammatory lesions associated with helminth infections or allergic conditions. In this review, Carolyn Behm and Karen Ovington outline some of the cellular and biological properties of eosinophils and evaluate the evidence for their role(s) in parasitic infections.     

Cell-mediated immune responses to sperm antigens: effects on mouse sperm and embryos

Sperm antigens were assessed for their ability to induce cell-mediated immune (CMI) responses. Purified fertilization antigen (FA-1), protamine, and the lithium diiodosalicylate (LIS)-solubilized sperm preparation activated presensitized lymphocytes to secrete soluble mediators that activated macrophages and significantly inhibited sperm motility and embryonic development. The FA-1, however, was the most potent antigen in inducing proliferative response as well as the release of soluble mediators. LIS-sperm preparation, which contained numerous antigens, showed the least activity. The unsensitized control spleen cells did not secrete any factor(s) when activated with the antigen. In conclusion, these results indicate that sperm antigens can specifically induce (CMI) factors that have detrimental effects on sperm motility and preimplantation embryos. These findings may have potential clinical implications for humans, especially in immunologic and unexplained infertility, recurrent abortions, and development of antisperm contraceptive vaccines.     

Antigen binding to lymphoid cells from unimmunized mice: IV. Shedding and reappearance of multiple antigen binding Ig receptors of T- and B-lymphocytes

Patterned antigen-binding cells (ABC) can bind two antigens and show “islands” of Ig receptor with mixed specificity. However, these cells, when unfixed, lose most of their bound fluorescent antigen within minutes upon warming above 0 °C. Residual antigen moves to one pole of the cell forming a “cap” within 5 min at room temperature. If such patterned ABC are capped with a single antigen, receptors to a second antigen can be detected on a portion of the capped cells, but only in the cap. The frequency of capped, “double” ABC approximated the frequency of patterned “double” ABC originally present.If lymphoid cells are mixed with fluorescent antigens at 0 °C and then incubated for 4 hr at 37 °, no ABC are found. When the cells are then fixed and the fluorescent antigens readded, new antigen-binding Ig receptors can be shown to have reappeared on the cell surface during the 4-hr incubation. The reappearance of antigen receptor could be inhibited by prior addition of either 10^?2M sodium azide or 50 μg/ml cycloheximide, implying that the receptors were actively synthesized by the cell. These inhibitors did not prevent shedding, but azide did inhibit the capping process. Both B-cells (bone marrow or spleen cells) and T-cells (splenic T-cells or 99.5% pure cortisone-resistant T-cells) were shown to regenerate multispecific ABC to the frequency found prior to incubation.     

O-methylated glycans from Toxocara are specific targets for antibody binding in human and animal infections

The parasitic helminth Toxocara canis is a widely distributed nematode of mammals. Larval parasites, which infect a wide range of hosts including mice and humans, export glycosylated macromolecules bearing novel methylated oligosaccharide structures, similar to the mammalian blood group antigen H but bearing one or two O-methylated substitutions on the terminal fucose and subterminal galactose residues. We have studied the reactivity of synthetic forms of these glycans to parasite-specific antibodies and mammalian immune system lectins. Murine antibodies, generated to T. canis infection, predominantly recognise the mono-O-methylated form with the beta-configuration of the GalNAc residue (MoMbeta), and antibodies are entirely IgM isotype. The mAb Tcn-2 reproduces this pattern, and shows little reactivity to either the alpha isomer (MoMalpha) or the di-O-methylated form (DiM). Antibodies generated to helminth infections other than T. canis were unreactive with the glycans, except antibodies to other members of the Toxocara genus. Hence, the carbohydrate structures represent immunogenic, genus-specific antigens. Antibodies from human toxocariasis patients are reactive with the same sugars, although preferentially towards DiM. Sera from unrelated helminth infections do not react, confirming the status of these structures as Toxocara-specific glycans. The human dendritic cell lectin, DC-SIGN, was found to bind both Toxocara excretory/secretory products and mammalian blood group antigen H3. However, DC-SIGN did not bind the synthetic glycans, indicating additional non-methylated carbohydrates may also play a role in the interaction between T. canis and its host.     

Transfer of osteosarcoma-specific cell-mediated immunity in hamsters by rabbit dialyzable leukocyte extracts

We have investigated the transfer of specific cell-mediated immunity (CMI) to osteosarcoma-associated antigens (OSAA) to hamsters with dialyzable leukocyte extracts (DLE) from OSAA-immunized rabbits. The transfer of specific CMI was determined by leukocyte adherence inhibition (LAI) assay and skin testing. DLE was prepared from rabbits immunized with OSAA, purified protein derivative (PPD), or fibrosarcoma cell plasma membrane preparation (FSM). Control DLE was prepared from rabbits injected with 0.85% NaCl. Significant leukocyte adherence inhibition was observed with leukocytes from hamsters that had received OSAA-specific, PPD-specific, and FSM-specific rabbit DLE, when OSAA, PPD, and FSM were used as antigens, respectively. Similarly, significant ear swelling after injection of OSAA, PPD, or FSM was observed only in hamsters that had received DLE from rabbits immunized with OSAA, PPD, or FSM, respectively. These results suggest that CMI specific for OSAA, PPD, or FSM can be transferred to normal hamsters by DLE from immunized rabbits.