The cytokinetics of monocytosis in acute salmonella infection in the rat.

The mechanisms responsible for monocytosis occurring in acute Salmonella infection were studied by means of isotopic labeling and autoradiography. Male (Lewis x BN)F(1) hybrid rats (160-180 g) were pulse-labeled with [(3)H]TdR at varying intervals with respect to the time of i.v. injection of about 10(6) living Salmonella enteritidis. The half time for monocytes in the blood was estimated from the exponential decline in the percentage of labeled monocytes. The average generation time for dividing monocyte precursors in bone marrow was estimated by fitting a regression line to the decline in median grain counts (halving-time = T(G)). After an initial fall, the absolute number of blood monocytes rose to a plateau about 2.5 x normal on day 5, suggesting the reimposition of steady state conditions. The half time of monocytes in the blood of infected rats was shortened to 25 h throughout the infection, compared with 61 h estimated in uninfected rats. T(G) was reduced to 15 h (days 1-3) but later reverted to the preinfection level of 34 h (days 4-8). Another early response to infection was the release of immature monocytes into the blood. These cells, however, were too few to offset the initial monocytopenia. Under these conditions, with little or no division of blood monocytes, the sustained monocytosis (days 4-8) must have been due to enlargement of the dividing precursor pool. Excessive loss of monocytes from the blood thus appears to activate a feedback mechanism. However, a more direct stimulating effect on monocyte production by endotoxin could have contributed substantially to the monocytosis.

Crossprotection against nontuberculous mycobacterial infections by Mycobacterium tuberculosis memory immune T lymphocytes.

Adoptive immunization of T cell-deficient recipient mice with M. tuberculosis-specific memory immune T lymphocytes conferred upon these animals the ability to express significantly enhanced resistance both to the homologous infection, and to three strains of nontuberculous mycobacteria. These results support the hypothesis, therefore, that antigenic determinants possessed by the four mycobacterial strains that are relevant to the generation of protective cellular immunity are identical or closely crossreactive.

The route of enteric infection in normal mice.

This study followed the early pathogenesis of orally induced murine typhoid fever. Intragastrically administered Salmonella enteritidis moves quickly through the normal undisturbed gut so that only a small residuum remains in the cecum and large intestine after the first few hours. Dye injection of the gut wall was used to show that lymph from discrete portions of the gastrointestinal tract drains to separate lymph nodes, probably via the regional Peyer's patches. Plating techniques capable of detecting a single colony-forming unit of S. enteritidis within the different Peyer's patches and draining lymph nodes indicate that, although the cecum and large intestine are exposed to large numbers of Salmonella for longer time periods than the small intestine, the primary site of bacterial penetration involves the distal ileum. This area of the small intestine as well as the cecum are both drained by the distal mesenteric lymph nodes, and were the only nodes which contained detectable numbers of viable Salmonella over the first 24 h of infection. Neither the pyloric nor the proximal mesenteric lymph nodes (which drain the stomach and duodenum) nor the pancreatic and caudal lymph nodes (which drain the transverse and descending colon) contained viable Salmonella. Salmonella were observed to infect the ileal mucosa and its Peyer's patches. With time, this infection progresses to the draining lymph node and ultimately reaches the liver and spleen. Some of the implications of these findings relative to the development of acquired resistance to enteric disease are discussed.

Protection against Mycobacterium tuberculosis infection by adoptive immunotherapy. Requirement for T cell-deficient recipients.

The results of this study demonstrate that spleen cells taken from mice at the height of the primary immune response to intravenous infection with Mycobacterium tuberculosis possess the capacity to transfer adoptive protection to M. tuberculosis-infected recipients, but only if these recipients are first rendered T cell-deficient, either by thymectomy and gamma irradiation, or by sublethal irradiation. A similar requirement was necessary to demonstrate the adoptive protection of the lungs after exposure to an acute aerosol-delivered M. tuberculosis infection. In both infectious models successful adoptive immunotherapy was shown to be mediated by T lymphocytes, which were acquired in the donor animals in response to the immunizing infection. It is proposed that the results of this study may serve as a basic model for the subsequent analysis of the nature of the T cell-mediated immune response to both systemic and aerogenic infections with M. tuberculosis.

Mechanisms of acquired resistance in mouse typhoid.

Experiments in vitro comparing normal mouse peritoneal macrophages with cells from Salmonella typhimurium-infected mice have shown that the "immune" macrophages have conspicuously enhanced microbicidal properties. Whereas normal macrophages could inactivate only 50 to 60% of intracellular S. typhimurium pretreated with immune serum, cells from infected animals killed virtually all ingested organisms and did so at an accelerated rate. Macrophages from Listeria monocytogenes-infected mice were shown to possess similarly enhanced microbicidal activity against S. typhimurium. Furthermore, the growth of S. typhimurium in the liver and spleen was more effectively restricted in Listeria-infected mice than in animals vaccinated with heat-killed S. typhimurium, even though the Listeria-infected animals possessed no demonstrable cross-reacting antibody to S. typhimurium. The lack of resistance in the mice vaccinated with heat-killed organisms could not be attributed to any deficiency of humoral factors, since the serum from these animals was as effective at promoting phagocytosis and killing by macrophages as serum from actively infected (and demonstrably resistant) mice. Conversely, Salmonella-infected mice were totally resistant to intravenous challenge with L. monocytogenes. The level of resistance in individual animals was related to the numbers of residual Salmonellae remaining in the tissues; mice with heavier residual infections being the more resistant. Specific antiserum from mice vaccinated with heat-killed S. typhimurium was found to be significantly protective only when the intraperitoneal route of challenge was employed. The foregoing studies have been interpreted to mean that enhancement of the microbicidal ability of macrophages is the mechanism of major importance in acquired resistance to S. typhimurium infection in mice.

Host-parasite relations in mouse typhoid.

The development of acquired resistance to Salmonella typhimurium has been studied in mice infected intravenously with small numbers of streptomycin-sensitive or streptomycin-resistant organisms. By the 14th day of a primary infection the mouse develops a mechanism capable of destroying completely a super infecting dose of organisms, but is unable to eliminate organisms of the primary infection. The latter are constantly returned to the circulation from necrotic foci at the sites of implantation. Passive transfer of serum from actively infected or vaccinated animals, and immunization with heat-killed organisms, increase the capacity of the host to clear organisms from the blood, but do not interfere to any significant extent with their subsequent multiplication in the tissues. It is concluded that the resistance of actively infected animals depends on a nonhumoral mechanism capable of destroying organisms from endogenous or exogenous sources.

Infection-immunity in experimental salmonellosis.

Salmonella enteritidis is highly virulent for the mouse causing an infection resembling mouse typhoid. Survivors of the infection are completely resistant to reinfection and eliminate a large challenge dose of virulent organisms within 72 hr. The antigenically related Salmonella gallinarum was almost avirulent for the mouse but animals vaccinated with this organism were equally capable of eliminating a lethal dose of virulent S. enteritidis. Living Salmonella pullorum, on the other hand, was quickly eliminated from the tissues of normal mice. Vaccination with this organism failed to evoke an effective bactericidal mechanism. Alcohol-killed vaccines of these three Salmonellae all produced an increase in blood clearance rate, but gave only marginal protection against S. enteritidis. Liver and spleen counts on these mice revealed a 1 to 2 day delay before any net increase in the total bacterial population could be observed. Immunization of mice with increasing doses of living Salmonella montevideo resulted in progressively greater killing of a challenge dose of S. enteritidis despite the absence of common somatic antigens between the two strains. The degree of protection varied with the size of the residual population of S. montevideo in the vaccinated mice. The significance of these findings in assessing the importance of various factors involved in the development of acquired resistance to Salmonella infections is discussed.

Mycobacterium bovis (BCG) infection of the lymph nodes of normal, immune, and cortisone-treated guinea pigs.

Strain-2 inbred guinea pigs were infected intradermally with 10(5)-10(7) viable BCG (Pasteur) organisms by means of multiple scarifications of shaven midflank skin. The spread of the BCG to the draining lymph nodes and on to the spleen was followed quantitatively for 28 days. The population of bacilli at the inoculation site increased as much as tenfold the first 14 days. The number of viable BCG organisms recovered from the primary draining superficial dorsal axillary and inguinal lymph nodes varied from 0.1 to 1.0% of the inoculum, with a further tenfold to 100-fold drop in counts for the secondary subclavian and lumbar lymph nodes. The bacterial counts for the various nodes increased substantially the first 14 days. By 28 days, as many as 1,000 viable bacilli were recovered from the spleen. Increasing the inoculum size or the number of inoculation sites increased the primary node counts and promoted a more extensive and rapid spread by the BCG population to the secondary lymph nodes and spleen. Prior vaccination of the host with living BCG decreased the spread of the BCG inoculum from the scarification site to the various draining lymph nodes. Multiple injections of cortisone tended to reverse this effect.

Effect of mycobacterium bovis (BCG) infection on the kinetics of the mononuclear cell response within the lung.

Specific pathogen-free LBN rats were parabiotically linked and the monocyte donor animal was labeled with multiple pulses of tritiated thymidine (1 microCi/g body weight). The right-hand (recipient) rat lungs were infected with 10(5) viable Mycobacterium bovis (BCG) Pasteur by the intravenous, aerogenic, or intratracheal routes. Control animals received heat-killed BCG or saline only, given intratracheally. The BCG infection resulted in a ten-fold increase in the number of heavily labeled, blood-derived monocytes recovered 24 hr later in the lung lavage fluid. The percentage of labeled cells peaked on day 3 and then declined slowly. Introduction of heat-killed BCG into the lung produced a smaller mononuclear cell influx but a marked polymorphonuclear phagocyte response that persisted for several days. The labeled monocyte counts for the infected recipient rat lung washouts were five to ten times those for the uninfected donor parabiont, except when the aerogenic infection route was used, when both donor and recipient rats were equally infected and both showed substantial increases in labeled monocytes in the lung washouts.

Effects of in vivo T lymphocyte subset depletion on mycobacterial infections in mice.

The relative importance of CD4+ and CD8+ T cell subsets in the expression of acquired resistance to systemic infection by Mycobacterium kansasii was determined. T cell subsets were depleted in thymectomized C57BL/6 mice by the intravenous administration of monoclonal antibodies directed against the relevant T cell determinants. Depletion of the CD4+ subset exacerbated the severity of the infection in intravenously challenged mice. This effect was apparent in the first 2 weeks of the infection and persisted throughout the 12 weeks of the study. On the other hand, depletion of the CD8+ cells had no apparent effect on the growth curves. Infections by Mycobacterium tuberculosis Erdman or bacille Calmette-Guérin (BCG) Pasteur were also substantially enhanced by CD4 depletion, but not by the depletion of CD8+ cells. The effect of subset depletion on infections by M. tuberculosis and BCG was examined in both innately susceptible C57BL/6 mice and innately resistant B6D2 mice.

Mycobacterial pathogenesis: a historical perspective.

Tuberculosis is an age-old human affliction which continues to flourish worldwide despite the development of effective drugs for its treatment and a vaccine (BCG) for its prevention. At least 8 million people die from this disease each year, a figure which is likely to increase as the AIDS epidemic continues its relentless spread into Africa and Southeast Asia. Consumption was shown to be caused by Mycobacterium tuberculosis more than a century ago, yet we still know very little about the mechanisms used by this organism to elude the normally effective cellular host defenses as it establishes a progressive infection within the lung. The majority of individuals exposed to tuberculous infection are able to limit the primary infection to the lungs and its lymph nodes, resulting in a latent form of the disease which can provide the host with a lifelong immunity to reinfection. While a great deal is known about the cellular mediators of this immune response (together with the cytokines which modulate them) we lack a clear understanding of the role that they play during the establishment of the dormant form of the disease. Live BCG vaccine has been widely used in many Third World countries as a major component of their tuberculosis control programs. However, several carefully controlled human trials have shown little protection achieved in vaccinated individuals. Development of improved vaccines, both for the prevention and therapy of this disease is an urgent research priority and a number of potential immunogens are under active investigation. However, our limited understanding of the pathogenesis of this chronic disease, together with a lack of data on the role played by different bacterial components in the modulation of the immune response, continues to severely limit our ability to develop a rational approach to this project. To achieve this goal, it will be necessary to establish innovative approaches to the presentation of protective antigens by taking advantage of recent advances in the molecular biology of this complex and enigmatic group of organisms.

Progressive pulmonary tuberculosis is not due to increasing numbers of viable bacilli in rabbits, mice and guinea pigs, but is due to a continuous host response to mycobacterial products.

Tuberculosis (TB) kills more people in the world today than any other infectious disease. A better vaccine to prevent clinical tuberculosis is greatly needed. Candidate vaccines are often evaluated by infecting rabbits, mice and guinea pigs by an aerosol of virulent tubercle bacilli and culturing their lungs for viable bacilli at various times thereafter. In all three species, however, the number of viable bacilli usually does not continuously increase until the host succumbs. The number of viable bacilli increases logarithmically for only about 3 weeks. Then, the host develops delayed-type hypersensitivity (DTH) and cell-mediated immunity (CMI), which keep the number of viable bacilli rather constant during the subsequent weeks. In the immunized host, DTH and CMI stop the logarithmic increase sooner than in the unimmunized controls, so that the stationary bacillary levels that follow are lower. This review analyzes host-parasite interactions in the lungs of rabbits, mice and guinea pigs. All three species cannot prevent inhaled fully virulent tubercle bacilli from establishing an infection, but they differ markedly in the type of the disease produced once it is established.

Cerebrovascular consequences of repeated exposure to NG-nitro-L-arginine methyl ester.

1. Acute treatment with the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) produces cerebral oligaemia. The effects of repeated exposure to L-NAME upon cerebral blood flow were examined to determine whether the enhanced NOS inhibition reported following chronic treatment might reduce cerebral perfusion to ischaemic levels. 2. Rats were treated with L-NAME (75 mg kg-1, i.p.) once daily for 10 days. Local cerebral blood flow and glucose utilization were measured by [14C]-iodoantipyrine and [14C]-2-deoxyglucose quantitative autoradiography respectively, either 1 h or 15 h after the last injection. A second group of rats was injected (i.p.) only once with L-NAME, either 1 h or 15 h prior to the measurement procedures. 3. Mean arterial blood pressure (MABP) was significantly increased (+35%) 1 h after a single injection of L-NAME. Although the hypertension was reduced 15 h after the injection (+13%), MABP remained significantly higher than control. 4. Local cerebral blood flow was significantly decreased 1 h after a single injection of L-NAME (ranging from -45% to -54%), and remained so even after 15 h (-39% to -48%). At neither time-point was there any change in glucose utilization. 5. At 15 h after the final injection of the chronic L-NAME treatment protocol, MABP was significantly elevated from control (+58%) and was also significantly higher than at 1 h following a single injection (+20%). There was no effect upon the established hypertension when rats treated chronically with L-NAME were challenged with a further injection of the drug and MABP was measured 1 h later, suggesting saturation of NOS inhibition. 6. Although reduced, cerebral blood flow was not significantly different from control when measured 15 h after the last injection of the chronic L-NAME treatment. When rats treated chronically with L-NAME were subjected to a further challenge with the drug, cerebral blood flow was reduced when measured 1 h after the acute injection (ranging from -34% to -41%). There was however evidence of some attenuation in the response when compared to that measured 1 h after a single injection of L-NAME (ranging from -45% to -54%). Thus, the cerebral circulation shows no evidence of either sustained L-NAME-induced vasoconstriction or saturated NOS inhibition following 10 daily injections of L-NAME. Chronic L-NAME treatment had no effect upon cerebral glucose use. 7. The trend towards re-establishment of cerebrovascular dilator tone and the normalization of cerebral flow-metabolism relationships could explain the lack of any ischaemic damage found in chronically treated rats, but the loss of an extended autoregulatory range afforded by acute L-NAME treatment may be responsible for an increased incidence of stroke.

Differential release of tumor necrosis factor-alpha from murine peritoneal macrophages stimulated with virulent and avirulent species of mycobacteria.

The ability of Mycobacterium tuberculosis H37Rv and H37Ra, M. bovis BCG and M. smegmatis to induce the secretion of tumor necrosis factor-alpha (TNF-alpha) by cultured murine peritoneal macrophages is inversely related to their virulence. The avirulent species of mycobacteria which were unable to persist in macrophages were capable of inducing significant levels of TNF-alpha compared to that formed in cultures infected with the virulent M. tuberculosis H37Rv. This difference was also associated with an inherent toxicity by live H37Rv for macrophage cultures. Heat-killed H37Rv was non-toxic and induced significant levels of TNF-alpha; in contrast, live and heat-killed suspensions of avirulent mycobacteria had an equivalent ability to trigger TNF-alpha secretion. The TNF-alpha response was dose-dependent, related directly to the percentage of infected cells, and peaked 6-12 h post-infection. An early and vigorous TNF-alpha response appears to be a marker of macrophage resistance, while the downregulation of this response seems associated with macrophage toxicity and unrestricted mycobacterial growth.

The immune response to mycobacterial infection: development of new vaccines.

Pulmonary tuberculosis continues to flourish worldwide despite our most vigorous attempts to control it. After nearly a century of study we still know very little about the virulence factors of M. tuberculosis of M. bovis or how they trigger the protective immune response within the infected host. This anti-tuberculous response is mediated by a population of specifically sensitised T lymphocytes which activate the monocytes entering the developing lesion from the bloodstream. The immunologically activated macrophage induces a persistent bacteriostasis which is usually sufficient to protect the host although it will not eliminate the infection altogether so that reactivation can occur whenever the cellular defences are depleted as a result of aging or immunosuppressive chemotherapy. Protective immunogens released by actively growing tubercle bacilli give rise to a protective cell-mediated, rather than a humoral (non-protective) immunity. The genes responsible for the production of these "protective" antigens are being cloned and transferred to suitable mycobacterial vectors by means of the newly developed "shuttle phasmid". Development of such recombinants constitute the first step in preparing more effective anti-tuberculous vaccines for future use against these important human and animal pathogens.

Effect of aztreonam on the growth of Pasteurella multocida in the lung.

Specific-pathogen-free ICR mice were infected aerogenically with Pasteurella multocida and, beginning 1 hour later, were treated with aztreonam (50 mg/kg of body weight). The number of viable bacilli in the lungs, liver, and spleen were determined at intervals for up to 36 hours. Aztreonam was bactericidal for growing bacilli in vitro and, when injected 1 and 5 hours after aerogenic exposure, provided greater than 80% protection after dosage at the level of 12.5 mg/kg. Below this dosage level, viable organisms persisted in the lungs and the spleen and many of the minimally treated mice eventually died of pasteurellosis. The survivors developed active immunity as a result of the continued sublethal infection. Aztreonam protects mice against an aerogenic infection with highly virulent P multocida and may be useful in the prevention and treatment of pasteurellosis in cattle.