Difference in antigen-presenting ability of macrophages between high- and low-responder mice in delayed-type hypersensitivity to Mycobacterium bovis BCG.

Purified protein derivative-pulsed spleen macrophages of Mycobacterium bovis BCG high-responder mice stimulated BCG-primed lymphocytes of F1 (low x high) mice well in vitro, but those of BCG low-responder mice did not.     

Immune responses to defined antigens of Mycobacterium bovis in cattle experimentally infected with Mycobacterium kansasii

Cross-reactive responses elicited by exposure to nontuberculous mycobacteria often confound the interpretation of antemortem tests for Mycobacterium bovis infection of cattle. The use of specific proteins (e.g., ESAT-6, CFP-10, and MPB83), however, generally enhances the specificity of bovine tuberculosis tests. While genes for these proteins are absent from many nontuberculous mycobacteria, they are present in M. kansasii. Instillation of M. kansasii into the tonsillar crypts of calves elicited delayed-type hypersensitivity and in vitro gamma interferon and nitrite concentration responses of leukocytes to M. avium and M. bovis purified protein derivatives (PPDs). While the responses of M. kansasii-inoculated calves to M. avium and M. bovis PPDs were approximately equivalent, the responses of M. bovis-inoculated calves to M. bovis PPD exceeded their respective responses to M. avium PPD. The gamma interferon and nitrite responses of M. kansasii-inoculated calves to recombinant ESAT-6-CFP-10 (rESAT-6-CFP-10) exceeded corresponding responses of noninoculated calves as early as 15 and 30 days after inoculation, respectively, and persisted throughout the study. The gamma interferon and nitrite responses of M. bovis-inoculated calves to rESAT-6-CFP-10 exceeded the corresponding responses of M. kansasii-inoculated calves beginning 30 days after inoculation. By using a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum antibodies were detected as early as 50 days after challenge with M. kansasii. By a multiantigen print immunoassay and immunoblotting, serum antibodies to MPB83, but not ESAT-6 or CFP-10, were detected in M. kansasii-inoculated calves; however, responses to MPB83 were notably weaker than those elicited by M. bovis infection. These findings indicate that M. kansasii infection of calves elicits specific responses that may confound the interpretation of bovine tuberculosis tests.     

Involvement of prostaglandin E1 in delayed-type hypersensitivity suppression induced with live Mycobacterium bovis BCG.

We previously showed the suppression of delayed-type hypersensitivity produced by live BCG-induced, plastic-adhering cells. The present study was undertaken to determine whether prostaglandin was one of the pharmacological mediators involved in this suppression. A high level of prostaglandin E1 was detected in the culture supernatant fluid of the live BCG-induced, adherent cells. Moreover, prostaglandin E1 could suppress the macrophage migration inhibition activity of peritoneal exudate cells from BCG cell wall-sensitized mice.     

Immune responses induced in cattle by vaccination with a recombinant adenovirus expressing Mycobacterial antigen 85A and Mycobacterium bovis BCG

Cattle were vaccinated with an adenovirus expressing the mycobacterial antigen 85A (rAd85A), with Mycobacterium bovis BCG followed by rAd85A heterologous boosting, or with rAd85A followed by BCG boosting. BCG/rAd85A resulted in the highest direct gamma interferon responses. Cultured enzyme-linked immunospot assay analysis demonstrated that memory responses were induced by all three protocols but were strongest after BCG/rAd85A and rAd85A/BCG vaccination.     

Tumour necrosis factor receptors and apoptosis of alveolar macrophages during early infection with attenuated and virulent Mycobacterium bovis

Apoptosis of macrophages has been reported as an effective host strategy to control the growth of intracellular pathogens, including pathogenic mycobacteria. Tumour necrosis factor-α (TNF-α) plays an important role in the modulation of apoptosis of infected macrophages. It exerts its biological activities via two distinct cell surface receptors, TNFR1 and TNFR2, whose extracellular domain can be released by proteolysis forming soluble TNF receptors (sTNFR1 and sTNFR2). The signalling through TNFR1 initiates the majority of the biological functions of TNF-α, leading to either cell death or survival whereas TNFR2 mediates primarily survival signals. Here, the expression of TNF-α receptors and the apoptosis of alveolar macrophages were investigated during the early phase of infection with attenuated and virulent mycobacteria in mice. A significant increase of apoptosis and high expression of TNFR1 were observed in alveolar macrophages at 3 and 7 days after infection with attenuated Mycobacterium bovis but only on day 7 in infection with the virulent M. bovis. Low surface expression of TNFR1 and increased levels of sTNFR1 on day 3 after infection by the virulent strain were associated with reduced rates of apoptotic macrophages. In addition, a significant reduction in apoptosis of alveolar macrophages was observed in TNFR1^−/− mice at day 3 after bacillus Calmette–Guérin infection. These results suggest a potential role for TNFR1 in mycobacteria-induced alveolar macrophage apoptosis in vivo. In this scenario, shedding of TNFR1 seems to contribute to the modulation of macrophage apoptosis in a strain-dependent manner.     

Enhancement of delayed-type hypersensitivity to Trypanosoma cruzi in mice treated with Mycobacterium bovis BCG and cyclophosphamide.

C57BL/10J mice treated with Mycobacterium bovis BCG and cyclophosphamide were immunized with disrupted epimastigotes or with living blood trypomastigotes from Trypanosoma cruzi and assayed for delayed hypersensitivity by footpad testing with epimastigote antigens. Enhanced and lasting reactions were observed in mice pretreated with BCG or cyclophosphamide or both and immunized with epimastigotes. Whereas BCG pretreatment clearly reduced the mortality rates of mice immunized with living blood forms, no enhancement of the delayed hypersensitivity responses was observed in animals treated with BCG or cyclophosphamide or both before infection. The production of high levels of delayed hypersensitivity in the absence of infection and its adoptive transfer with cells could help to evaluate the participation of cell-mediated immunity in the protection against T. cruzi.     

Differential expression of gamma interferon mRNA induced by attenuated and virulent Mycobacterium tuberculosis in guinea pig cells after Mycobacterium bovis BCG vaccination

To determine whether Mycobacterium bovis BCG vaccination would alter gamma interferon (IFN-gamma) mRNA expression in guinea pig cells exposed to Mycobacterium tuberculosis, we cloned a cDNA encoding guinea pig IFN-gamma from a spleen cell cDNA library. The cDNA is composed of 1,110 bp, with an open reading frame encoding a 166-amino-acid protein which shows 56 and 41% amino acid sequence homology to human and mouse IFN-gamma, respectively. Spleen or lymph node cells from na?ve and BCG-vaccinated guinea pigs were stimulated with purified protein derivative (PPD) or M. tuberculosis H37Ra or H37Rv, and the total RNA was subjected to Northern blot analysis with a (32)P-labeled probe derived from the cDNA clone. Compared to the IFN-gamma mRNA expression in cells of na?ve animals, that in spleen and lymph node cells exposed to various stimuli was enhanced after BCG vaccination. However, there was a significant reduction in IFN-gamma mRNA levels when cells were stimulated with a multiplicity of infection of greater than 1 virulent M. tuberculosis bacterium per 10 cells. The enhanced IFN-gamma mRNA response in BCG-vaccinated animals was associated with an increase in the proportions of CD4(+) T cells in the spleens, as determined by fluorescence-activated cell sorter analysis. Furthermore, the nonadherent population in the spleens enriched either by panning with anti-guinea pig immunoglobulin G-coated plates or by purification on nylon wool columns produced more IFN-gamma mRNA than whole spleen cells following stimulation with concanavalin A or PPD. This indicates that T cells are principally responsible for the upregulation of IFN-gamma mRNA expression following BCG vaccination. The mechanism by which virulent mycobacteria suppress IFN-gamma mRNA accumulation is currently under investigation.     

Induction of suppressor T cells in delayed-type hypersensitivity to Mycobacterium bovis BCG in low-responder mice.

The induction of delayed-type hypersensitivity to Mycobacterium bovis BCG was specifically inhibited by suppressor T cells in C3H/He, a strain of mice which is a low responder to BCG. The existence of these suppressor cells was confirmed by an adoptive transfer of spleen cells of BCG-injected mice into cyclophosphamide-treated recipients. The suppressor cells appeared in the spleens of the mice 2 to 7 days after intravenous BCG injection. They were sensitive to anti-theta serum and complement and did not adhere to Sephadex G-10. A pretreatment of the mice with cyclophosphamide eliminated the suppression of delayed-type hypersensitivity. These suppressor cells effectively inhibited the induction of delayed-type hypersensitivity to BCG, but showed only weak effect on the expression of it.     

Early antibody responses to experimental Mycobacterium bovis infection of cattle

Bovine tuberculosis persists as a costly zoonotic disease in numerous countries despite extensive eradication and control efforts. Sequential serum samples obtained from Mycobacterium bovis-infected cattle were evaluated for seroreactivity to mycobacterial antigens. Animals received M. bovis by aerosol, intratonsil, intranasal, or intratracheal inoculation. Assays included the multiantigen print immunoassay for determination of antigen recognition patterns, immunoblot analysis for sensitive kinetic studies, and the VetTB STAT-PAK test, a novel, rapid test based on lateral-flow technology. Responses to MPB83 were detected for all M. bovis-infected animals regardless of the route or strain of M. bovis used for inoculation. Other less commonly recognized antigens included ESAT-6, CFP-10, and MPB70. Responses to MPB83 were detectable as early as 4 weeks after inoculation, were boosted upon injection of purified protein derivatives for skin testing, and persisted throughout the course of each of the four challenge studies. MPB83-specific immunoglobulin M (IgM) was detected prior to MPB83-specific IgG detection; however, early IgM responses rapidly waned, suggesting a benefit of tests that detect both IgM- and IgG-specific antibodies. The VetTB STAT-PAK test detected responses in sera from 60% (15/25) of the animals by 7 weeks after challenge and detected responses in 96% (24/25) of the animals by 18 weeks. These findings demonstrate the potential for new-generation antibody-based tests for the early detection of M. bovis infection in cattle.     

Cytotoxic T-cell responses to Mycobacterium bovis during experimental infection of cattle with bovine tuberculosis

Cytotoxic T-cell responses are thought to play a significant role in the host defence against mycobacterial infections. Little is understood about such responses of cattle to Mycobacterium bovis, the causative agent of bovine tuberculosis. The work described in this report demonstrates the activity of cytotoxic cells during experimental infection of cattle with M. bovis. The cytotoxic cells were found to have the ability to specifically lyse macrophages infected with M. bovis and were detected in peripheral blood lymphocytes after in vitro re-exposure to M. bovis. Cytotoxic activity was detected 4 weeks after experimental infection with M. bovis; a similar level of activity was maintained during the infection and it was mediated by both WC1+gammadelta and CD8+ T cells. In addition, inhibition of the growth of M. bovis within infected macrophages was detected when they were exposed to cultures containing M. bovis-specific cytotoxic cells. The ability to detect cytotoxic cells after infection of cattle with M. bovis will allow their activity to be measured during vaccination trials. Correlation of cytotoxic activity with disease outcome may aid in the design of new vaccines and vaccination strategies.     

Environmental strains of Mycobacterium avium interfere with immune responses associated with Mycobacterium bovis BCG vaccination

Prior exposure of a vaccinee to certain species of environmental mycobacteria can prime the immune system against common mycobacterial antigens, which can in turn reduce the subsequent efficacy of live attenuated mycobacterial vaccines (such as Mycobacterium bovis BCG), in both human and livestock vaccination programs. In this study, two strains of Mycobacterium avium, both isolated from New Zealand livestock, were investigated to determine their growth characteristics and effects on the immune system in murine models. Markedly different effects on the immune system were observed; an IS901-negative strain (WAg 207) induced significant up-regulation of cell surface activation markers (major histocompatibility complex II, CD80, and CD86) on in vitro-derived dendritic cells and induced the release of proinflammatory monokines (interleukin-1beta [IL-1beta], IL-6, and tumor necrosis factor alpha) in dendritic cell-macrophage cocultures following direct in vitro contact of cells with bacteria. In contrast, an IS901-positive strain (WAg 206) had none of these effects. When mice were exposed to M. avium via oral infection prior to BCG parenteral immunization, both strains were shown to be capable of decreasing subsequent antigen-stimulated gamma interferon secretion by splenic lymphocytes, although this effect was more significant for strain WAg 206. Both strains also induced a mycobacterial antigen-specific serological response in M. avium-sensitized and BCG-immunized mice; this response was greater in WAg 206-sensitized mice, and there was a predominance of immunoglobulin G1 antibody. The down-regulation of IFN-gamma responses and the up-regulation of antibody responses are characteristic of a switch to a type 2 immune response. The different results may be linked to the inherent growth characteristics of the two strains, since WAg 206 was shown to grow slowly in murine macrophages in vitro and to cause a persistent systemic infection following infection in vivo, while WAg 207 grew fast and did not persist in mice. The implications of these findings for BCG vaccination protocols are discussed.     

Differentiation by molecular typing of Mycobacterium bovis strains causing tuberculosis in cattle and goats.

Forty Mycobacterium bovis isolates from cattle and goats were analyzed by using different repetitive genetic markers. The 23 M. bovis strains from goats were found to carry six to eight copies of the insertion sequence IS6110. In contrast, most of the bovine isolates contained only a single copy of this element. The standardized IS6110 fingerprinting by restriction fragment length polymorphism (RFLP), described for Mycobacterium tuberculosis strains, allowed the differentiation of caprine strains. Although this method was not useful for typing bovine isolates, the repetitive elements pTBN12 and DR proved to be suitable for this purpose. A procedure using PCR which amplifies IS6110 in the outward direction was found to be as sensitive as RFLP for typing M. bovis strains from goats. The use of PCR and RFLP methods based on the IS6110 polymorphism would be useful for epidemiological studies of caprine tuberculosis. The results are consistent with different strains of M. bovis being implicated in bovine and caprine tuberculosis.     

P-selectin directs T lymphocyte-mediated injury in delayed-type hypersensitivity responses: studies in glomerulonephritis and cutaneous delayed-type hypersensitivity

The role of P-selectin in T lymphocyte accumulation and injury was studied in delayed-type hypersensitivity (DTH) responses in the skin and glomeruli of rats. Sprague Dawley rats were sensitized to sheep globulin and challenged 5 days later in the skin by subcutaneous injection and simultaneously in glomeruli by intravenous injection of a subnephritogenic dose of sheep anti-rat glomerular basement membrane globulin. This resulted in cutaneous and glomerular T lymphocyte-dependent macrophage influx and injury characteristic of DTH. Up-regulation of P-selectin expression on endothelial cells was observed in both inflammatory lesions. Treatment of rats with anti-CD5 antibody immediately prior to antigen challenge prevented the development of injury as assessed by measurement of proteinuria and skin swelling, as well as local T cell and macrophage accumulation in the glomerulus and in the skin, but did not block up-regulation endothelial cell P-selectin. Treatment with anti-CD4 antibody produced similar results. Blocking P-selectin in vivo with a functionally inhibitory antibody prevented development of proteinuria and skin swelling following antigen challenge. Local accumulation of T cells and macrophages was markedly attenuated in glomeruli and the skin and up-regulation of endothelial cell P-selectin was prevented. These data demonstrate that P-selectin is locally up-regulated on endothelial cells in T cell-dependent glomerular and cutaneous inflammation and suggests a pivotal functional role for P-selectin in local T cell recruitment and subsequent injury in DTH.     

Induction of delayed-type hypersensitivity responses to PPD: dendritic cells in synergy with 5-hydroxytryptamine can substitute for macrophages.

We describe the use of 5-hydroxytryptamine (5HT) as an adjuvant in the induction of the delayed-type hypersensitivity (DTH) response to purified protein derivative (PPD). Based upon our previous studies with antigen-pulsed macrophages (M phi), we have shown that both the Day 2 early (2 hr) reaction and the Day 3 (24 hr) reaction are augmented if 5HT is incorporated into the priming injection. Furthermore, we have confirmed that in contrast to M phi, antigen-pulsed dendritic cells (DC) fail to prime the early (2 hr) component of DTH. However, DC do prime the early response if injected along with 5HT. A peripheral 5HT antagonist, ICS 205-930, inhibits both the M phi-mediated and the 5HT/DC-primed reactions. These findings support the hypothesis that DTH reactions require a cascade of both inflammatory and immunological signals, and that in mice vascular permeability mediated via 5HT is important in the early phase of the reaction.     

Differing role of dendritic cells and macrophages in the induction of delayed-type hypersensitivity responses to PPD.

In order to study the antigen-presenting cell (APC) requirements for the priming of delayed-type hypersensitivity (DTH), murine spleen cells were fractionated on bovine serum albumin gradients, pulsed in vitro with tuberculin, and then injected subcutaneously into normal mice. The other footpad was challenged with tuberculin between 24 hr and 7 days later and swelling was measured 2 hr and 18-24 hr after challenge. Optimum priming for 2-hr and 24-hr responses at 7 days was achieved by an injection of 5 X 10(5) tuberculin-pulsed intermediate-density Fc + ve cells. Time-course studies revealed that the 2-hr component could be elicited as early as 24 hr after injection of pulsed APC, while the 24-hr component became significant at 3 days. Elimination of T cells or B cells did not affect the response. Injection of pulsed APC into allogeneic mice primed the 2-hr but not the 24-hr component. Neither pulsed high-density cells (mostly T cells) nor pulsed dendritic cells (DC) primed mice for these responses. Failure to elicit DTH after injection of tuberculin-pulsed DC was due to their failure to prime the 2-hr component, which other authors have shown to be a prerequisite for the appearance of the later components. That DC did prime the MHC-restricted 24-hr component was demonstrated by protocols involving the use of both macrophages and DC as APC.     

Generation of attenuated Mycobacterium bovis strains by signature-tagged mutagenesis for discovery of novel vaccine candidates

Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, has a particularly wide host range and causes tuberculosis in most mammals, including humans. A signature tag mutagenesis approach, which employed illegitimate recombination and infection of guinea pigs, was applied to M. bovis to discover genes important for virulence and to find potential vaccine candidates. Fifteen attenuated mutants were identified, four of which produced no lesions when inoculated separately into guinea pigs. One of these four mutants had nine deleted genes including mmpL4 and sigK and, in guinea pigs with aerosol challenge, provided protection against tuberculosis at least equal to that of M. bovis BCG. Seven mutants had mutations near the esxA (esat-6) locus, and immunoblot analysis of these confirmed the essential role of other genes at this locus in the secretion of EsxA (ESAT-6) and EsxB (CFP10). Mutations in the eight other attenuated mutants were widely spread through the chromosome and included pks1, which is naturally inactivated in clinical strains of M. tuberculosis. Many genes identified were different from those found by signature tag mutagenesis of M. tuberculosis by use of a mouse infection model and illustrate how the use of different approaches enables identification of a wider range of attenuating mutants.     

The replication of virulent and attenuated strains of African swine fever virus in porcine macrophages

Summary The replication of virulent and attenuated strains of African swine fever virus (ASFV) was studied in pure cultures of swine macrophages. To ensure complete destruction of the macrophage monolayers about 50–100 times more virulent ASFV was needed than attenuated virus although both isolates could be used to establish persistently infected cultures.Interferon did not appear to influence virus yields from such cultures.Fluorescent and electron microscopy studies of infected macrophages suggested that the cycle of infection of the two isolates was different.With 3 Figures     

Suppression of the delayed-type hypersensitivity and cell-mediated immune responses to Listeria monocytogenes induced by Pseudomonas aeruginosa.

Pseudomonas aeruginosa-mediated suppression of the immune response to Listeria monocytogenes was investigated in mice. Because delayed-type hypersensitivity (DTH) footpad swelling to L. monocytogenes was suppressed equally in lipopolysaccharide-responsive and -hyporesponsive mouse strains, the lipopolysaccharide component of P. aeruginosa could not have been the suppressive agent. Mucoid P. aeruginosa cells were no more suppressive than their nonmucoid revertants; therefore, mucoid coating was not an additional immunosuppressive element. Interleukin-1 and macrophage inhibitory factor production to L. monocytogenes and clearance of L. monocytogenes from mouse spleens were all decreased by prior Pseudomonas infection, indicating that cell-mediated immunity, as well as DTH, was decreased to a sublethal Listeria dose. The timing of Pseudomonas exposure relative to Listeria sensitization was varied. P. aeruginosa injected 24 or 6 h before or at the same time as L. monocytogenes depressed DTH to Listeria challenge 7 days later. Animals treated in this way could not respond to reinfection with L. monocytogenes at 13 days. P. aeruginosa administered to L. monocytogenes-sensitized mice at the time of footpad challenge was suppressive, but these mice responded normally upon reinfection. It appears that P. aeruginosa induced two types of suppression to L. monocytogenes: a transient suppression, affecting DTH challenge but not resensitization, and a longer lasting suppression that did not permit mice exposed to P. aeruginosa at the time of Listeria sensitization to respond to subsequent Listeria exposure.     

Induction of delayed-type hypersensitivity to Mycobacterium leprae in healthy individuals.

Delayed-type hypersensitivity (DTH) to a soluble Mycobacterium leprae skin test antigen (SML) was successfully induced in healthy volunteers following immunization with 2 X 10(8) killed armadillo-derived M. leprae. No better sensitization was obtained by a mixture of live BCG and killed M. leprae. The relative specificity of the DTH reaction to SML has been demonstrated in this study, since little cross-reactivity was observed to PPD, after immunization with BCG ro M, leprae alone, or combined. Moreover, armadillo-derived M. leprae readily induced a specific hypersensitivity with the time course DTH response associated with protective immunity suggesting that this bacterial preparation may be a candidate for an effective anti-leprosy vaccine.