Resistance and susceptibility to infection in inbred murine strains. II. Variations in the effect of treatment with thymosin fraction 5 on the release of lymphokines in vivo

Of nine inbred murine strains sensitized intravenously with killed lyophilized Candida albicans and challenged 3 weeks later with a C. albicans filtrate, four strains were low responders and five were high responders in the in vivo release of migration inhibitory factor (MIF) and gamma interferon (IFN-gamma). An identical distribution of high- and low-responder strains occurred in response to sensitization with Mycobacterium bovis BCG and subsequent challenge with old tuberculin. Treatment of the murine strains with thymosin fraction 5 prior to sensitization and challenge had different effects: (a) the high-responder strains had a decrease in their release in vivo of the two lymphokines; (b) three of five of the low-responder strains had a striking increase in the in vivo release of MIF and IFN-gamma; and (c) one low-responder strain did not have its response altered. A parallelism existed between the capacity of a murine strain to release the two lymphokines in vivo on stimulation with C. albicans antigens and the capacity of that strain to resist intravenous infection with living C. albicans.     

The stability and immunogenicity of a dispersed-grown freeze-dried Pasteur BCG vaccine

The level of antituberculous immunity seems to be related to the number of memory T cells induced. This may vary as a function of the multiplication and persistence of BCG in host tissues. The most important requirements for a BCG vaccine are, therefore, the immunogenicity of the strain, the high proportion of live to dead bacilli, and adequate dispersion and low levels of soluble antigens. The surface-grown Pasteur BCG vaccine contains a very high proportion of bacilli killed by ball-milling and freeze-drying. It also contains clumps and soluble antigens, all factors influencing cell-mediated immune processes and viability control. Therefore, several batches of vaccine were prepared on an industrial scale using one of the most immunogenic strains (French 1173 P2) and grown as dispersed bacilli by a modified cell type culture method. This method provided fully viable, well-dispersed vaccines which have a viability and heat stability superior to that of the classical surface-grown BCG. The immunogenicity was checked by multiplication and persistence in mouse organs and the skin reactivity and tuberculin hypersensitivity in guinea-pigs showed results comparable to those obtained with classical vaccine. Small-scale tests in children showed superior immunogenicity of the dispersed as opposed to the classical vaccine and there was no suppurative adenitis.     

Mechanisms in the in vivo release of lymphokines. 1. Comparative kinetics in the release of six lymphokines in inbred strains of mice

Lymphokines were detected in the sera of 16 strains of inbred mice sensitized intravenously with cell walls of Mycobacterium bovis strain BCG and challenged subsequently intravenously with old tuberculin (OT). Variations occurred between the strains in the types and quantities of six lymphokines studied, namely, chemotactic factor (CF), type II interferon (IF II), lymphotoxin (LT), migration inhibitory factor (MIF), mitogenic factor (MF), and skin reactive factor (SRF). The times for maximum release of the lymphokines in the different strains were similar for MIF, IF II, SRF, and MF, but differed for CF and LT. The degree of activity of MIF and IF II generally paralleled one another in the different strains but such parallelism did not occur for the other four lymphokines. Each of the 16 strains was a high responder for at least one of the lymphokines, indicating that in sensitized mice the release or presence in the circulation of lymphokines in response to a specific antigen is a selective process. Thus, each strain may have an individual combination of lymphokines, interactions of which may determine the types of pathway utilized in an immunological response.     

The role played the lipid fractions of Mycobacterium bovis BCG in the development of antituberculosis immunity in an animal experiment.

The authors investigated the antituberculosis and antitumour immunogenicity as well as tuberculin allergenicity of the lipid fractions from Mycobacterium bovis BCG strains of Danish, French, Japanese origin and of Czechoslovak 725. The fractions explored included phospholipids, Cord factor, ethanol-extractable lipids, waxes A, B, C + D and fats. The fractions were divided into three groups according to their effectiveness. 1. The Cord factor and phospholipids from all the studied strains were effective in the antituberculosis and antitumour models with the only exception of strain 725 phospholipids. Phospholipids from all strains were capable of inducing tuberculin allergy. 2. In the second group (waxes A, C + D and lipids extractable by ethanol) a variance was observed in the antigenic properties of identical fractions from different strains suggesting differing metabolism in the strains producing these fractions. A mixture of waxes C + D from the French and Danish strains showed a degree of suppression in its antituberculosis effectiveness. 3. Waxes B and fats were entirely ineffective in the antitumour model and, with the exception of waxes B from strain 725 and fats from the Japanese strain, in the antituberculosis model. The antituberculosis and antitumour effectiveness directly depended on the content of a mycolic acid complex in fractions. Tuberculin allergenicity was associated with the intensity of phospholipid production by mycobacteria.     

Refractoriness to migration inhibitory factor of macrophages of LPS nonresponder mouse strains.

The responsiveness to macrophage migration inhibitory factor (MIF) of peritoneal exudate cells (PEC) from the LPS unresponsive C3H/HeJ and C57BL/10ScCR mice was assessed by the indirect agarose microdroplet macrophage migration inhibition assay. No migration inhibition with PEC from C3H/HeJ nor C57BL/10ScCR mice was detected, whereas PEC from both C3H/HeN and C57BL/10Sn mice were significantly inhibited by even a 1/32 dilution of MIF-containing supernatants. Responsiveness to MIF of C3H/HeJ PEC could, however, be induced. In vivo inoculations of Mycobacterium bovis, strain BCG, 7 days before in vitro assay rendered C3H/HeJ PEC responsive to MIF. The lack of responsiveness to MIF by C3H/HeJ PEC appeared related to some form of suppression, since a mixture of PEC from C3H/HeN mice with 10 to 15% PEC from C3H/HeJ mice resulted in undetectable migration inhibition at any MIF dilution. In contrast to the usual lack of responsiveness of their macrophage to MIF, C3H/HeJ mice were able to produce MIK in response to PPD as well as their counterpart C3H/HeN mice after BCG sensitization. These results demonstrate that macrophages from C3H/HeJ and C57BL/10ScCR mice are unable to be inhibited in their in vitro migration of MIF (possibly being directly or indirectly influenced by a suppressor cell), whereas lymphoid cells from at least one of these strains, the C3H/HeJ mice, can produce MIF in response to antigenic stimulation.     

Delayed hypersensitivity and nonspecific cellular immunity. The conditions determining the maximally expressed immunity activity

In vivo experiments on the infection of mice with influenza A virus and Francisella tularensis and in vitro experiments on the bactericidal activity of macrophages have demonstrated the conditions leading to the maximally pronounced activation of immunity by means of preparations inducing delayed hypersensitivity (DH). The following conditions have been determined: the presence of pronounced DH previously to the injection of old tuberculin (OT) and staphylococcal phagolysate (SP) used as challenge antigens, the specificity and peculiar features of the antigenic structure of the challenge agent, the time of its administration after the course of multiple sensitizing injections of BCG and staphylococci, the dosage of OT and SP and the scheme of their administration, the desirability of their local use. The time of the maximum activation of cell-mediated immunity after the injection of OT and SP to sensitized animals with a high level of DH and the duration of such activation have been established.     

Properties of TAS-1D3, a Tuberculin-Active Substance from BCG,in Regard to Delayed Hypersensitivity

The properties of TAS-1D3, a tuberculin-active substance purified from the cell extract of Mycobacterium bovis BCG, were studied in vivo and in vitro. In the delayed hypersensitivity skin reaction, TAS-1D3 showed far more potent activity than tuberculin purified protein derivative (PPD) in guinea pigs sensitized with BCG vaccine. This was consistently observed from 6 to 24 weeks after sensitization. The histological findings of the skin reaction to TAS-1D3 were similar to those of the reaction to PPD. Moreover, TAS-1D3 induced well both thymidine incorporation and the production of migration inhibitory factor (MIF) by the spleen cells from guinea pigs sensitized with BCG vaccine. In contrast, TAS-1D3 showed weaker activity than PPD in guinea pigs sensitized with either heat-killed M. tuberculosis Aoyama B or heat-killed M. tuberculosis H37Ra, and it weakly stimulated the spleen cells from animals sensitized with M. tuberculosis Aoyama B to incorporate thymidine and to produce MIF.     

Induction of macrophage migration inhibition factor-producing cells in vitro

The in vitro induction of effector cells mediating delayed type hypersensitivity (DTH) responses was demonstrated during joint cultivation of plastic non-adherent spleen cells of intact CBA mice and adherent spleen cells pretreated with MBSA or OVA. The activity of these effector cells was studied by testing their ability to produce MIF after stimulation by a specific antigen. The formation of MIF-producing cells was observed with the use of low antigen doses (about 50 micrograms/ml). High OVA doses (250 micrograms/ml) were found to suppress the development of MIF-producing cells. MIF-producing cells obtained after either in vitro or in vivo exposure to identical antigens did not differ in their activity.     

Immune response to BCG-Moreau (Rio de Janeiro) strain. Spectrum of delayed hypersensitivity in genetically defined mice

Generation of delayed hypersensitivity (DTH) in genetically defined mice immunized with Mycobacterium bovis BCG of the Moreau (Rio de Janeiro) strain was studied. This vaccine strain has been reported as the most virulent and able to induce strong tuberculin sensitivity. Mice were selected by the expression of Bcg gene trait, by responsiveness to mycobacterial antigens and H2 haplotype. DTH was evaluated by the ear-swelling test of mice immunized subcutaneously with live BCG at doses ranging from 1 microgram to 1000 micrograms. A survey of inbred strains of mice showed H2b and H2q mice as high responders, H2d as an intermediate responder, H2k as a low responder and H2a as a non-responder. Study of H2-congenic pairs of high and non-responder strains showed significant DTH in all mice independently of the genetic background and H2 haplotype. A mouse strain expressing Bcg (r) trait displayed DTH superior to a Bcg (s) strain. Comparison of DTH response of strains expressing Bcg (r) or (s) trait showed no relationship between the Bcg locus and DTH to mycobacteria. These data suggest DTH is under polygenic control including the major histocompatibility complex but excluding the Bcg locus.     

Delayed Hypersensitivity in Relation to Suppression of Growth of Listeria monocytogenes by Guinea Pig Macrophages

Prototypes of delayed hypersensitivity (tuberculin allergy, graft rejection immunity, and contact dermatitis) were established in guinea pigs. The macrophages from peritoneal exudates of such animals were examined for their capacities to suppress the growth of Listeria monocytogenes in vitro. Only the macrophages from animals sensitized to BCG clearly exhibited this property.     

Leukocyte procoagulant activity in man: an in vitro correlate of delayed-type hypersensitivity

Human mononuclear leukocytes generate cell-bound procoagulant activity (LPCA) after incubation with an antigen (mumps or tuberculin) to which the donor was previously sensitized. An inhibitor of coagulation appears to be liberated into the extracellular culture fluid during incubation of leukocytes with the sensitizing antigen. Removal of this activity before measuring LPCA resulted in a reliable test that correlated directly with delayed skin reactivity. The assay was particularly sensitive in that cells from weakly sensitized donors who reacted only to high doses of tuberculin (100 TU) in the delayed skin tests produced detectable LPCA in vitro. By contrast cells from weakly sensitized donors did not react to PPD in the lymphocyte blast transformation test or the direct macrophage migration inhibition factor test. The LPCA assay correlated closely with the blast transformation and MIF tests in which cells were used from more strongly sensitized donors who reacted in skin tests with lower doses of tuberculin (1 or 10 TU). The assays were antigen-specific in that cells from donors sensitive to mumps antigen but not to tuberculin reacted only to mumps antigen in vitro. The assay was extremely reproducible; cells from individual donors reacted to the same extent over a period of 8 mo). We propose that the assay system reported here represents an improved method for the measurement of cell-mediated immunity in vitro because it requires fewer donor cells, is technically simpler, and is more sensitive than previously described methods.     

Immune response to BCG-Moreau (Rio de Janeiro) strain. Spectrum of delayed hypersensitivity in genetically defined mice

Abstract Generation of delayed hypersensitivity (DTH) in genetically defined mice immunized with Mycobacterium bovis BCG of the Moreau (Rio de Janeiro) strain was studied. This vaccine strain has been reported as the most virulent and able to induce strong tuberculin sensitivity. Mice were selected by the expression of Bcg gene trait, by responsiveness to mycobacterial antigens and H2 haplotype. DTH was evaluated by the ear-swelling test of mice immunized subcutaneously with live BCG at doses ranging from 1 μg to 1000 μg. A survey of inbred strains of mice showed H2b and H2q mice as high responders, H2d as an intermediate responder, H2k as a low responder and H2a as a non-responder. Study of H2-congenic pairs of high and non-responder strains showed significant DTH in all mice independently of the genetic background and H2 haplotype. A mouse strain expressing Bcg (r) trait displayed DTH superior to a Bcg (s) strain. Comparison of DTH response of strains expressing Bcg (r) or (s) trait showed no relationship between the Bcg locus and DTH to mycobacteria. These data suggest DTH is under polygenic control including the major histocompatibility complex but excluding the Bcg locus.     

Resistance and susceptibility to infection in inbred murine strains. IV. Effects of dietary zinc

Mice of several inbred strains have been fed diets containing either large amounts of zinc (300 ppm Zn), small amounts of zinc (5 ppm Zn), or routine laboratory mouse chow. When the mice are fed on a high-zinc diet, murine strains, such as C3H/HeJ, AKR/J, and CBA/CaJ, which are normally susceptible to infection with Candida albicans and which normally release low titers of migration-inhibition factor (MIF) in vivo into the circulation, become more resistant to infection with C. albicans and release higher titers of MIF in vivo into the circulation. In addition, their capacity to elicit delayed type hypersensitivity responses may be enhanced. When the mice are maintained on a low-zinc diet, murine strains, such as C57Bl/10SNJ, which are normally resistant to infection with C. albicans and which normally release high titers of MIF in vivo into the circulation on appropriate antigenic challenge, become more susceptible to infection and release lower titers of MIF into the circulation. Under these conditions of low-zinc concentrations in the diet, their capacity to elicit delayed type hypersensitivity may be reduced. Thus, the concentration of zinc in the diet may have a pronounced effect on some in vivo parameters of cell-mediated immunity.     

Delayed hypersensitivity and nonspecific cellular immunity. The role of mono- and polynuclear phagocytes

Differences in the influence produced by sensitization with BCG vaccine and Staphylococcus aureus and by the reaction of delayed hypersensitivity (DH) induced, respectively, by the injection of old tuberculin and staphylococcal phagolysate on the phagocytic activity of peritoneal macrophages and blood leukocytes in different animals were experimentally demonstrated. A considerable activation of the bactericidal and ingesting functions of macrophages was observed in animals showing a pronounced DH reaction (rabbits, guinea pigs and mice), while in Wistar rats no such activation was noted. The latter showed no DH reaction after sensitization with BCG vaccine and the injection of the specific antigen. Among different strains of mice, the activation of macrophages occurred in the animals with the most pronounced DH reaction. Sensitization with BCG vaccine led to an insignificant sensitization of macrophages, and sensitization with S. aureus even suppressed the phagocytic activity of macrophages. The treatment of mice with antimacrophagal preparations (carrageenan, silica and trypan blue, but T-lymphocyte antiserum) before and after the injection of the specific antigen into the sensitized animals abolished the stimulation of anti-infection immunity.     

Effect of delayed hypersensitivity due to Staphylococcus or BCG vaccine on the sensitivity of animals to infection with heterologous bacteria or influenza virus

Experiments on mice of different strains have demonstrated that sensitization with BCG vaccine slightly increases resistance to infection with Francisella tularensis, Escherichia coli 819 and influenza A2 virus in mice of those strains which are capable of developing a high level of delayed hypersensitivity (DH). On the contrary, sensitization with Staphylococcus aureus b-243 decreases this resistance. A sharp increase in resistance to infection has been achieved in sensitized animals receiving DH-inducing specific antigen (old tuberculin or staphylococcal phagolysate) 24 hours before inoculation. This increased resistance to infection is due mainly to the eliminating capacity of the reticuloendothelial system and not to the bactericidal factors of the serum. The level of sensitization and the manifestation of DH reaction have been found to be genetically determined and to govern the degree of activation of nonspecific immunity.     

19F NMR measurements of NO production in hypertensive ISIAH and OXYS rats

Recently we demonstrated the principal possibility of application of 19F NMR spin-trapping technique for in vivo *NO detection [Free Radic. Biol. Med. 36 (2004) 248]. In the present study, we employed this method to elucidate the significance of *NO availability in animal models of hypertension. In vivo *NO-induced conversion of the hydroxylamine of the fluorinated nitronyl nitroxide (HNN) to the hydroxylamine of the iminonitroxide (HIN) in hypertensive ISIAH and OXYS rat strains and normotensive Wistar rat strain was measured. Significantly lower HIN/HNN ratios were measured in the blood of the hypertensive rats. The NMR data were found to positively correlate with the levels of nitrite/nitrate evaluated by Griess method and negatively correlate with the blood pressure. In comparison with other traditionally used methods 19F NMR spectroscopy allows in vivo evaluation of *NO production and provides the basis for in vivo *NO imaging.     

Infection with Bacillus Calmette-Guérin activates murine thymus-independent (B) lymphocytes.

Thymus-independent (B) lymphocytes from BCG-infected mice respond with enhanced or depressed levels of proliferation relative to the nonspecific levels of stimulation induced by tuberculin. The type of response obtained depends on how BCG was administered and the source of the lymphocytes. The proliferation of splenic B lymphocytes was depressed when BCG was given i.v. and unchanged when BCG was given subcutaneously, whereas the lymph node B cell response was enhanced regardless of the route of injecting BCG. BCG was found also to be a mitogen for normal B cells in vitro and to stimulate polyclonal activation of B cells in vivo. Finally, evidence is presented which indicates that the depressed splenic B cell response to tuberculin may be due to the activation of suppressor T cells.     

Autoluminescent Mycobacterium tuberculosis for rapid, real-time, non-invasive assessment of drug and vaccine efficacy

Preclinical efforts to discover and develop new drugs and vaccines for tuberculosis are hampered by the reliance on colony-forming unit (CFU) counts as primary outcomes for in vivo efficacy studies and the slow growth of Mycobacterium tuberculosis. The utility of bioluminescent M. tuberculosis reporter strains for real-time in vitro and ex vivo assessment of drug and vaccine activity has been demonstrated but a simple, non-invasive, real-time surrogate marker to replace CFU counts for real-time evaluation of drug and vaccine efficacy in vivo has not been described. We describe the development of a fully virulent and stable autoluminescent strain of M. tuberculosis and proof-of-concept experiments demonstrating its utility for in vivo bioluminescence imaging to assess the efficacy of new drugs and vaccines for tuberculosis in a mouse model. Relative light unit (RLU) counts paralleled CFU counts during the active phase of bacterial growth, with a lower limit of detection of approximately 10(6) CFU in live, anesthetized mice. Experiments distinguishing active from inactive anti-tuberculosis drugs and bacteriostatic drug effects from bactericidal effects were completed in less than 5 days. The ability of a recombinant BCG vaccine to limit bacterial growth was demonstrated within 3 weeks. Use of this autoluminescent reporter strain has the potential to drastically reduce the time, effort, animals and costs consumed in the evaluation of drug activity in vitro and the in vivo assessment of drug and vaccine efficacy.     

The fbpA/sapM double knock out strain of Mycobacterium tuberculosis is highly attenuated and immunogenic in macrophages

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death due to bacterial infections in mankind, and BCG, an attenuated strain of Mycobacterium bovis, is an approved vaccine. BCG sequesters in immature phagosomes of antigen presenting cells (APCs), which do not fuse with lysosomes, leading to decreased antigen processing and reduced Th1 responses. However, an Mtb derived ΔfbpA attenuated mutant underwent limited phagosome maturation, enhanced immunogenicity and was as effective as BCG in protecting mice against TB. To facilitate phagosome maturation of ΔfbpA, we disrupted an additional gene sapM, which encodes for an acid phosphatase. Compared to the wild type Mtb, the ΔfbpAΔsapM (double knock out; DKO) strain was attenuated for growth in mouse macrophages and PMA activated human THP1 macrophages. Attenuation correlated with increased oxidants in macrophages in response to DKO infection and enhanced labeling of lysosomal markers (CD63 and rab7) on DKO phagosomes. An in vitro Antigen 85B peptide presentation assay was used to determine antigen presentation to T cells by APCs infected with DKO or other mycobacterial strains. This revealed that DKO infected APCs showed the strongest ability to present Ag85B to T cells (>2500 pgs/mL in 4 hrs) as compared to APCs infected with wild type Mtb or ΔfbpA or ΔsapM strain (<1000 pgs/mL in 4 hrs), indicating that DKO strain has enhanced immunogenicity than other strains. The ability of DKO to undergo lysosomal fusion and vacuolar acidification correlated with antigen presentation since bafilomycin, that inhibits acidification in APCs, reduced antigen presentation. Finally, the DKO vaccine elicited a better Th1 response in mice after subcutaneous vaccination than either ΔfbpA or ΔsapM. Since ΔfbpA has been used in mice as a candidate vaccine and the DKO (ΔfbpAΔsapM) mutant is more immunogenic than ΔfbpA, we propose the DKO is a potential anti-tuberculosis vaccine.     

Decreased capacity of recombinant 45/47-kDa molecules (Apa) of Mycobacterium tuberculosis to stimulate T lymphocyte responses related to changes in their mannosylation pattern.

The Apa molecules secreted by Mycobacterium tuberculosis, Mycobacterium bovis, or BCG have been identified as major immunodominant antigens. Mass spectrometry analysis indicated similar mannosylation, a complete pattern from 1 up to 9 hexose residues/mole of protein, of the native species from the 3 reference strains. The recombinant antigen expressed in M. smegmatis revealed a different mannosylation pattern: species containing 7 to 9 sugar residues/mole of protein were in the highest proportion, whereas species bearing a low number of sugar residues were almost absent. The 45/47-kDa recombinant antigen expressed in E. coli was devoid of sugar residues. The proteins purified from M. tuberculosis, M. bovis, or BCG have a high capacity to elicit in vivo potent delayed-type hypersensitivity (DTH) reactions and to stimulate in vitro sensitized T lymphocytes of guinea pigs immunized with living BCG. The recombinant Apa expressed in Mycobacterium smegmatis was 4-fold less potent in vivo in the DTH assay and 10-fold less active in vitro to stimulate sensitized T lymphocytes than the native proteins. The recombinant protein expressed in Escherichia coli was nearly unable to elicit DTH reactions in vivo or to stimulate T lymphocytes in vitro. Thus the observed biological effects were related to the extent of glycosylation of the antigen.