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To screen effective antigens as therapeutic subunit vaccines against Mycobacterium latent infection, we did bioinformatics analysis and literature review to identify effective antigens and evaluated the immunogenicity of five antigens highly expressed in dormant bacteria, which included Rv2031c (HspX), Rv2626c (Hrp1), Rv2007c (FdxA), Rv1738 and Rv3130c. Then, several fusion proteins such as Rv2007c-Rv2626c (F6), Rv2031c-Rv1738-Rv1733c (H83), ESAT6-Rv1738-Rv2626c (LT40), ESAT6-Ag85B-MPT64 <190-198> -Mtb8.4 (EAMM), and EAMM-Rv2626c (LT70) were constructed and their therapeutic effects were evaluated in pulmonary Mycobacterium bovis Bacilli Calmette-Guérin (BCG) - latently infected rabbit or mouse models. The results showed that EAMM and F6 plus H83 had therapeutic effect against BCG latent infection in the rabbit model, respectively, and that the combination of EAMM with F6 plus H83 significantly reduced the bacterial load. In addition, the fusion proteins LT40 and LT70 consisting of multistage antigens showed promising therapeutic effects in the mouse model. We conclude that subunit vaccines consisting of both latency and replicating-associated antigens show promising therapeutic effects in BCG latent infection animal models., (© 2017 The Foundation for the Scandinavian Journal of Immunology.)

Background: BCG protection is suboptimal and there is significant interest to develop new tuberculosis (TB) vaccines. However, there are significant limitations of the current vaccine evaluation systems in the mouse model. Here, we developed a BCG-challenge rabbit skin model as a new way to evaluate the protective efficacy of selected TB subunit vaccine candidates. Methods: Rabbits were immunized with subunit vaccines, including EAMM (ESAT6-Ag85B-MPT64 <190-198> -Mtb8.4), MH (Mtb10.4-HspX), and LT70 (ESAT6-Ag85B-MPT64 <190-198> -Mtb8.4-Rv2626c) three times subcutaneously every 3-weeks and challenged with the attenuated Mycobacterium bovis BCG intradermally 6-weeks after last immunization. The immune response induced by the vaccine candidates was measured, the histopathology induced by the BCG challenge was studied, and the number of bacilli in the liquefied caseum was determined. Results: The subunit vaccines generated high antigen-specific IgG antibodies and fastened the liquefaction and healing process, and significantly reduced the viable BCG load. The subunit vaccine LT70 and EAMM-MH reduced BCG bacterial load in comparison to proteins EAMM, MH, Rv2626c, and also BCG itself. The Koch phenomena induced by the LT70 and combination of EAMM-MH were the same as that produced by BCG itself and were more rapid than those induced by the other proteins and the saline controls. Conclusions: The subunit vaccines LT70 and the combination of EAMM-MH showed promising protective efficacy as expected in the rabbit skin model, which can serve as a visual and convenient new model for evaluating TB vaccines.

This review hopes to improve the selection of new tuberculosis (TB) vaccines by providing several perspectives on the immunization of humans, mice, guinea pigs, rabbits, and monkeys which have not usually been considered. (i) In human TB vaccine trials, the low rate of healing of Mycobacterium bovis BCG lesions (used as the control group) would distinguish individuals who might be helped by vaccination from the 95% who do not need it and would make these trials more conclusive. (ii) The rabbit immune response to Mycobacterium tuberculosis is much more effective in arresting tuberculosis than those of other laboratory animals, so pulmonary tubercle counting in rabbits should be included in all preclinical TB vaccine testing. (iii) Both delayed-type hypersensitivity (DTH) and cell-mediated immunity (CMI) are necessary to control the growth of M. tuberculosis. The testing of new TB vaccines in mice or in guinea pigs may not detect important antigens needed for human immunization. Mice respond poorly to tuberculin-like antigens that cause DTH. Guinea pigs respond poorly to antigens that cause CMI. Rabbits and humans respond well to both DTH and CMI antigens. Since monkeys are very susceptible to M. tuberculosis, they may not be as useful as rabbits for preclinical vaccine evaluation. (iv) Critical antigens (possibly ESAT-6 or CFP-10) might increase the immunity of the host to a greater extent than that produced by a natural M. tuberculosis infection and therefore would be useful in both prophylaxis and immunotherapy. Such critical antigens would increase the host's ability to neutralize key components of M. tuberculosis that enable it to survive in both laboratory animals and humans.

To control tuberculosis in the world today an additional approach would be most welcomed. Preventing (or reducing) pulmonary cavity formation is one such approach that has been almost completely neglected. Pulmonary cavity formation and the extracellular growth of tubercle bacilli in cavities cause bronchial spread of the disease in adult patients and spread of the bacillus to the environment where they infect other people. Therefore, cavity formation perpetuates tuberculosis in mankind. If no cavities form, the patient is much less infectious. Also, cavity formation often allows the tubercle bacillus to multiply (extracellularly) to tremendous numbers. Therefore, in humans almost all multidrug-resistant tubercle bacilli develop in cavities. This communication reviews the literature on liquefaction and cavity formation, and lists some of the responsible hydrolytic enzymes. It also describes a simple method to identify inhibitory pharmaceuticals, i.e., to observe their effect on the liquefaction and ulceration of skin lesions produced in rabbits by ascending concentrations of live or dead tubercle bacilli.

The large reservoir of human latent tuberculosis (TB) contributes to the global success of the pathogen, Mycobacterium tuberculosis (Mtb). We sought to test whether aerosol infection of rabbits with Mtb H37Rv could model paucibacillary human latent TB. The lung burden of infection peaked at 5 weeks after aerosol infection followed by host containment of infection that was achieved in all rabbits. One-third of rabbits had at least one caseous granuloma with culturable bacilli at 36 weeks after infection suggesting persistent paucibacillary infection. Corticosteroid-induced immunosuppression initiated after disease containment resulted in reactivation of disease. Seventy-two percent of rabbits had culturable bacilli in the right upper lung lobe homogenates compared to none of the untreated controls. Discontinuation of dexamethasone led to predictable lymphoid recovery, with a proportion of rabbits developing multicentric large caseous granuloma. The development and severity of the immune reconstitution inflammatory syndrome (IRIS) was dependent on the antigen load at the time of immunosuppression and subsequent bacillary replication during corticosteroid-induced immunosuppression. Clinically, many aspects were similar to IRIS in severely immunosuppressed HIV-infected patients who have functional restoration of T cells in response to effective (highly active) antiretroviral therapy. This corticosteroid model is the only animal model of the IRIS. Further study of the rabbit model of TB latency, reactivation and IRIS may be important in understanding the immunopathogenesis of these poorly modeled states as well as for improved diagnostics for specific stages of disease.

We sought to characterize the lung cellular immune responses to inhaled Mycobacterium tuberculosis (Mtb) of the susceptible inbred Thorbecke rabbit (the genomically sequenced strain, now unavailable) and compare it to outbred, Mtb-resistant, New Zealand White rabbits. Using Mtb CDC1551, we confirmed that the inbred rabbits allowed establishment of infection with this low virulence strain, compared to poor establishment in outbred rabbits. With a more virulent strain, Mtb Erdman, that establishes infection well in both rabbit strains, we analyzed granulomas from rabbit lungs 5 weeks after aerosol infection. The lung granulomas of inbred rabbits had significantly higher frequencies of cells expressing MHC Class II and CD11b, and lower frequencies of CD8+ T cells than the outbred controls. Macrophage-sized cells expressing MHC Class II in inbred rabbit granulomas showed significantly decreased intensity of expression, suggesting impaired maturation. Although the inbred dermal tuberculin reactions were decreased, the in vitro IFN-gamma mRNA responses of hilar node lymphocytes to tuberculin were higher than those of outbred rabbits. Further delineation of the outbred rabbit's resistant immune response to Mtb infection is warranted.

Malnutrition is common in the developing world, where tuberculosis is often endemic. Rabbits infected with aerosolized Mycobacterium tuberculosis that subsequently became inadvertently and transiently malnourished had compromised cell-mediated immunity comparable to that of the rabbits immunosuppressed with dexamethasone. They had significant leukopenia and reduced delayed-type hypersensitivity responses. Malnutrition dampened cell-mediated immunity and would interfere with diagnostic tests that rely on intact CD4 T-cell responses.

The rabbit model of tuberculosis (TB) is important because rabbits develop a disease that is similar to TB in humans, namely, granulomas with caseous necrosis, liquefaction, and cavities. We describe here a comparison of inbred and outbred New Zealand White rabbits infected by aerosol with either Mycobacterium tuberculosis Erdman or H37Rv strain. Five weeks after infection with either bacillary strain, the inbred rabbits had significantly larger pulmonary tubercles than did outbred rabbits (2.7 versus 1.4 mm in diameter; P < 0.01). After infection with H37Rv, the inbred rabbits had significantly more pulmonary tubercles than did the outbred rabbits (98 +/- 12 versus 33 +/- 13; P < 0.01), with more mycobacterial CFU per tubercle (809 +/- 210 versus 215 +/- 115; P = 0.027) (means +/- standard errors of the means). Compared with histologic examination of lung granulomas from outbred rabbits, histologic examination of those from inbred rabbits showed more caseous necrosis, more visible bacilli, and fewer mature epithelioid cells. The delayed-type hypersensitivity (DTH) responses to intradermal tuberculin were significantly lower, and peritoneal macrophages from uninfected inbred rabbits produced significantly less tumor necrosis factor alpha after lipopolysaccharide (LPS) stimulation in vitro than those from the outbred rabbits (2,413 +/- 1,154 versus 8,879 +/- 966 pg/ml). We conclude that these inbred rabbits were more susceptible to TB than their outbred counterparts and had an impaired ability to contain disease, resulting in more grossly visible tubercles that were larger than those observed in outbred rabbits. Preliminary evidence is presented for a cell-mediated immune defect with lower DTH responses and macrophages that have a decreased ability to respond to in vitro stimulation with LPS or M. tuberculosis infection.

The rabbit model of tuberculosis has been used historically to differentiate between Mycobacterium tuberculosis and Mycobacterium bovis based on their relative virulence in this animal host. M. tuberculosis infection in market rabbits is cleared over time, whereas infection with M. bovis results in chronic, progressive, cavitary disease leading to death. Because of the innate resistance of commercial rabbits to M. tuberculosis, 320 to 1,890 log-phase, actively growing inhaled bacilli were required to form one grossly visible pulmonary tubercle at 5 weeks. The range of inhaled doses required to make one tubercle allows us to determine the relative pathogenicities of different strains. Fewer inhaled organisms of the M. tuberculosis Erdman strain were required than of M. tuberculosis H37Rv to produce a visible lesion at 5 weeks. Furthermore, with the Erdman strain, only 7 of 15 rabbits had healed lesions at 16 to 18 weeks; among the other animals, two had chronic, progressive cavitary disease, a phenotype usually seen only with M. bovis infection. Genotypic investigation of the Erdman strain with an H37Rv-based microarray identified gene differences in the RD6 region. Southern blot and PCR structural genetic analysis showed significant differences between M. tuberculosis strains in this region. Correlation of the relative pathogenicity, including disease severity, in the rabbit model with the strain genotype may help identify stage-specific M. tuberculosis genes important in human disease.

This review is a synthesis and analysis of our nine experimental pathology papers on macrophage kinetics in dermal tuberculous lesions produced in rabbits by BCG. It is presented at this time to summarize the macrophage kinetics in both active and essentially healed tuberculous lesions and to suggest that the bacilli frequently multiply and are destroyed in the viable granulation tissue of many small arrested tuberculous lesions. The turnover of mononuclear cells (MN)-which were mostly macrophages with some medium and large lymphocytes-was most rapid in BCG lesions at 2-3 weeks (when tuberculin sensitivity and acquired cellular resistance were at their peaks). At this time, more macrophages entered, more died or left, more remained at the site, and more became activated than before or afterwards. Before this time, the host had no delayed-type hypersensitivity (DTH) and cell-mediated immunity (CMI), so that no antigen-specific enhancement of the inflammatory response occurred. After this time, the bacilli and their antigenic products had decreased, so that the stimuli for cell infiltration and activation were reduced. In "healed" lesions, the MN turnover still occurred, but was decreased. The continuous entry of live non-activated macrophages into the viable parts of tuberculous lesions provides fresh intracellular sites where tubercle bacilli may multiply before they are again inhibited by the DTH and CMI of the host. In tuberculosis, bacillary dormancy of long duration may only be present in caseous necrotic tissue where no live host cells exist.

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., (Copyright 2001 Harcourt Publishers Ltd.)

A major milestone in tuberculosis research occurred in June 1998 with the report of the genomic sequence of Mycobacterium tuberculosis H37Rv. The complete determination of the 4411529 base pairs of the M. tuberculosis genome opens avenues for new scientific opportunities in basic science and clinical research on tuberculosis. In this paper we will review the findings presented by the complete genome and discuss the impact that this sequence will have on the areas of comparative genomics, bacterial pathogenesis, and diagnostics development for tuberculosis. Indirect benefits of the complete genome sequence are anticipated in the areas of drug development and vaccine development as future discoveries in bacterial pathogenesis and immunology accrue.

The virulence of the CDC1551 strain of Mycobacterium tuberculosis was compared to that of H37Rv in a rabbit inhalation model. While rabbits that inhaled the two strains produced equal numbers of grossly visible primary tubercles, CDC1551 tubercles were smaller and contained fewer bacilli than H37Rv tubercles. These findings suggest that a miniepidemic near the Kentucky-Tennessee border caused by CDC1551 was due not to increased virulence but to increased transmissibility.

This report elucidates four aspects of the immunology of pulmonary tuberculosis produced in rabbits: (i) the virulence of bovine-type tubercle bacilli, strain Ravenel S, (ii) systemic factors influencing the generation of visible primary pulmonary tubercles, (iii) differences in tuberculin sensitivity of rabbits and humans, and (iv) the effect of Mycobacterium vaccae immunotherapy on cavitary tuberculosis. Laboratory strain Ravenel S (ATCC 35720) was not fully virulent. Fully virulent strains produce one visible primary pulmonary tubercle for each three bacillary units inhaled. Strain ATCC 35720 produced one such tubercle for each 18 to 107 bacillary units inhaled, indicating that its virulence was reduced by 6- to 36-fold. When a low dose of this Ravenel S strain was inhaled, the host resistance (measured by the number of inhaled bacilli needed to generate one visible primary pulmonary tubercle) was increased at least 3.5-fold compared to the host resistance when a high dose was inhaled. Rabbits and humans differ in the degree and in the maintenance of their dermal sensitivities to tuberculin. Compared to rabbits, humans are 100 times more sensitive to tuberculin. Also, at 33 weeks rabbits with well-controlled cavitary tuberculosis usually showed a decrease in their tuberculin reactions of about 50% from peak values, whereas humans with such well-controlled tuberculosis are thought to maintain strong reactions for many years. These species differences may be due to desensitization to group II mycobacterial antigens in the rabbits because they have a different diet and a different type of digestive tract. M. vaccae immunotherapy of rabbits with cavitary tuberculosis produced no statistically significant effects. Experiments with many more rabbits would be required to prove whether or not such immunotherapy is beneficial.

To our knowledge, this is the first sequential study of cytokines in tissue sections of developing and healing tuberculous (BCG) lesions. In situ hybridization, immunohistochemical, and RT-PCR techniques were used. Cytokine mRNAs showed a biphasic pattern. The percentage of mononuclear cells (MN) containing IL-1beta, TNF-alpha, MCP-1, and IL-8 mRNAs was highest in 1- to 3-day lesions, apparently because of the nonspecific inflammatory response caused by the tubercle bacilli in the BCG vaccine. At 5 days, this percentage was significantly reduced. With IFN-gamma, the peak and trough were delayed by 2 days. By 9 days, the percentage of MN containing the mRNAs of all five cytokines had again increased and the rabbits had become tuberculin-positive. In general, MCP-1 and TNF-alpha proteins and the vascular adhesion molecules, ICAM, VCAM, and perhaps ELAM, peaked at about 3 days. Many mononuclear cells surrounding the central areas of solid and liquefied caseous necrosis contained chemokine IL-8 mRNA. IL-8 is known to attract PMN, and PMN were present nearby. In contrast, MN containing chemokine MCP-1 mRNA were present more peripherally in areas rich in macrophages and lymphocytes. The early nonspecific cytokine response seems to be an adjuvant effect of the mycobacteria in BCG vaccine in that it causes a rapid entry of macrophages, lymphocytes, granulocytes, and probably dendritic cells into local sites of antigen deposition. This effect should be considered in developing improved vaccines for the prevention of tuberculosis, because BCG vaccines producing a strong early cytokine response should be more immunogenic than BCG vaccines with similar antigens producing a weak response.

Development, peak and healing lesions were induced in the skin of rabbits by topical applications (on different days) of the chemical irritant sulfur mustard (SM). Immediately after the rabbits were euthanized, the intact lesions were excised and organ-cultured for 17 to 20 hours. The culture fluids from early, peak and healing SM lesions all showed high chemotactic activity for both PMN and MN. This finding suggests that the PMN and MN, seen microscopically in tissue sections of the lesions, were entering continuously, even during the healing process. The chemotaxins identified were the eicosanoid LTB4, the chemokine IL-8, and proteases producing the complement fragment C5a. Other studies from our laboratory showed that the number of cells containing IL-1, IL-8, MCP-1, and GRO mRNAs was increased in SM lesions. Chemotactic activity was released by both live and dead (frozen and thawed) cell suspensions of PMN, MN, and fibroblasts, suggesting that these cells were major sources of the chemotaxins produced by the SM lesion explants. Explants of normal skin produced considerable chemotactic activity for MN, but not for PMN. Chemotactic activity for PMN, and the release of LTB4, IL-8 and proteases cleaving C5 to C5a, occurred only in explants infiltrated by leukocytes.

Activation of the microvasculature is a major component of the inflammatory response. During inflammation the vascular endothelium not only becomes more permeable to plasma proteins but also develops adhesion molecules that initiate the local immigration of leukocytes. We describe herein the in vivo changes in the three major vascular adhesion molecules during the development and healing of two types of rabbit dermal inflammatory lesions: (1) acute lesions produced in rabbits by the topical application of 1% sulfur mustard (SM, the military irritant/toxicant); and (2) chronic (immune-mediated) lesions produced in rabbits by intradermal injections of Mycobacterium bovis (BCG), the vaccine strain of tubercle bacillus. In each case, frozen tissue sections were made from lesions of various ages and stained immunohistochemically for von Willebrand (vW) factor to measure the total functional microvasculature. The sections were also stained immunohistochemically for the vascular endothelial adhesion molecules ICAM-1, ELAM-1 (E-selectin), and VCAM-1, and for the leukocyte ligands for ICAM-1: LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18). Infiltrating monocytes and lymphocytes expressed the LFA-1 ligand and infiltrating PMN expressed the MAC-1 ligand. The area of stained microvasculature per square millimeter of tissue section was determined with the use of a computerized image analyzer. Edema and cell infiltration spread apart the microvessels, changing the number of microvessels per square millimeter of tissue section. Three methods of assessing such changes are presented. In SM lesions, endothelial ICAM levels were decreased from normal by about 50% at 1 and 2 days (when the lesions reached their peak size) and returned to normal at 3 and 6 days (during the healing process). ELAM rose in peak SM lesions and remained high during healing. VCAM levels, however, were only elevated in the 6-day (almost healed) lesions. In BCG lesions the levels of endothelial ICAM and VCAM (and to a lesser extent ELAM) were increased at 9 days and remained so as the size of the lesions peaked at 23 days. During the healing phase at 37 days, the elevated ICAM and VCAM levels decreased but the slightly increased ELAM levels persisted. These findings indicate that ELAM plays a major role in acute inflammation and that VCAM and ICAM play major roles in chronic inflammation. VCAM is known to be monocyte and lymphocyte selective.

Liquefaction of solid caseous tuberculous lesions and the subsequent cavity formation are probably the most dangerous processes in the pathogenesis of human pulmonary tuberculosis. In liquefied caseum, the tubercle bacilli grow extracellularly for the first time since the onset of the disease and can reach such large numbers that mutants with antimicrobial resistance may develop. From a cavity, the bacilli enter the bronchial tree and spread to other parts of the lung and also to other people. Of the commonly used laboratory animals, the rabbit is the only one in which cavitary tuberculosis can be readily produced. This report is the first to describe and analyze the complete course of cavitary tuberculosis, produced by aerosolized virulent bovine-type tubercle bacilli in commercially available New Zealand white rabbits. After the inhalation of 220 to 880 bacillary units, all of the rabbits were overtly well until they were sacrificed at 33 weeks. After the inhalation of 3,900 to 5,800 bacillary units, half of the rabbits died of progressive tuberculosis between 5 and 9 weeks and the other half lived until they were sacrificed at 18 weeks. Pulmonary cavities developed in both low- and high-dose groups, some beginning as early as 6 weeks. Bacilli from primary cavities sometimes caused nearby secondary cavities, but more frequently, they ascended the bronchial escalator, were swallowed, and caused secondary tubercles in the lymphoid tissue of the appendix and ileocecal junction. Histologically, and by culture, the number of bacilli found in the liquefied caseum varied from many to comparatively few. Strong tuberculin reactions at 4 weeks after infection were associated with fewer primary lesions, while strong tuberculin reactions at 33 weeks were associated with more cavitary lesions. In the tuberculous granulation tissue surrounding caseous and liquefied pulmonary foci and cavities, we found many mature epithelioid macrophages that contained high levels of the proteinase cathepsin D. Therefore, cathepsin D probably plays a major role in the liquefaction of solid caseous material and in the subsequent cavity formation.

Developing and healing dermal inflammatory lesions were produced in rabbits by the topical application of dilute sulfur mustard (SM), the military vesicant. In tissue sections of such lesions, cells containing the mRNA of important cytokines were identified with in situ hybridization techniques. These cytokines were neutrophil attractant/activation protein-1 (NAP-1 (also called IL-8), monocyte chemoattractant (activating) protein 1 (MCP-1), interleukin 1 (beta) (IL-1 (beta)), and GRO (a growth factor and chemokine). Mononuclear cells (mainly macrophages and activated fibroblasts) contained the mRNA of all four of these cytokines. A higher percentage of cytokine-producing mononuclear cells (macrophages and activated fibroblasts) was present in lesions at 2 days (their peak size) than at 6 days, when they were almost healed. Granulocytes emigrated from the bloodstream, passed through the lesions, and were the major constituent of the protective crust. This sequence correlated with the distribution of cells able to produce NAP-1: At 2 days and 6 days, the mononuclears that contained messenger RNA for this granulocyte chemoattractant were found mainly in the upper part of the dermis. At 2 days and 6 days, cells containing the mRNA of IL-1, a primary cytokine, were also found predominantly in the upper dermis, i.e., nearest the site of injury. In contrast, mononuclears containing the mRNA of MCP-1 (a monocyte chemoattractant), and the mRNA of GRO (a granulocyte chemoattractant) were more equally distributed throughout the dermis. SM stimulated hair follicle epithelial cells to up-regulate GRO mRNA and, to a lesser degree, NAP-1 mRNA. Apparently, the irritation produced by SM directly or indirectly induces such epithelial cells to manufacture these growth factors. In the rabbit, hair follicles are known to be the main source of new epithelial cells after the covering epithelium has been destroyed. Therefore, GRO is probably a major autocrine-paracrine stimulus for such repair. A brief review of the role of cytokines in dermal inflammation is presented.

The tubercle bacillus is a facultative intracellular parasite that grows well in non-activated macrophages. When large numbers of these bacilli have grown intracellularly within such macrophages, a cytotoxic immune response, herein called tissue-damaging (or necrotizing) delayed-type hypersensitivity (DTH), kills the macrophages (and usually some of the surrounding tissue), forming the caseous center of the developing tubercle. In solid caseum, tubercle bacilli may survive, but do not multiply. When bacilli escape from the edge of the caseum, they are rapidly ingested by nearby viable macrophages. If these macrophages have not been activated, the bacilli again multiply intracellularly, and the cytotoxic immune response kills the bacilli-laden macrophages (and surrounding tissue), thus enlarging the caseous center. In hosts that develop poor activation of macrophages, this process is repeated until much of the lung is destroyed. In hosts that can develop good activation of macrophages (by cytokines from antigen-specific T cells), herein called cell-mediated immunity (CMI), the caseous centers become surrounded by these activated macrophages, which ingest and destroy the bacilli escaping from the caseum. This process can arrest the disease. Unfortunately, the caseous center may liquefy in such resistant hosts. In the liquefied menstruum, the bacilli may grow extracellularly (for the first time during the course of the disease), reaching tremendous numbers. The cytotoxic immune response to these numerous bacilli and their tuberculin-like products causes much tissue necrosis, including erosion of the walls of small bronchi, which results in cavity formation. From such cavities, the bacilli spread to other parts of the lung and to the environment. The extracellular multiplication of tubercle bacilli in the liquefied caseum is the main reason why tuberculosis perpetuates itself in mankind. It is also the reason why antimicrobial drug-resistant bacillary strains develop. To elucidate the various mechanisms involved in macrophage activation, caseation, and liquefaction is a major challenge for tuberculosis researchers today.

The production of H2O2 by cells in cold paraformaldehyde-fixed frozen sections of inflammatory lesions was histochemically demonstrated by incubating them with diaminobenzidine (DAB) for 2 to 6 h. Catalase (150 micrograms/ml, about 1400 U/ml) inhibited the reaction, indicating that H2O2 was required to produce the chromogenic DAB product. Granulocytes (PMNs and eosinophils) were the main types of cells stained by the DAB reaction. Positive staining of macrophages was less frequent. The H2O2 was produced by metabolic enzymes that were still active after cell death and mild fixation. An atmosphere of 95 to 100% oxygen enhanced the specific DAB reaction, and an atmosphere of 100% nitrogen eliminated it. The DAB histochemical reaction to detect H2O2 requires the presence of peroxidases to produce the colored reaction product. Within our tissue sections, such peroxidases were evidently present in excess, because addition of low concentrations of H2O2 significantly increased the reaction product. Although some of the H2O2 produced by the granulocytes may have been derived from the dismutation of superoxide (O2-), the NADPH oxidase pathway for O2- formation did not seem to be involved: NADPH oxidase, a rather labile enzyme, should not be active after mild fixation, and diphenyleneiodonium (100 microM), an inhibitor of flavine-requiring NADPH oxidase, did not inhibit the reaction. Reactive nitrogen intermediates were also not involved, because NG-monomethyl-L-arginine and NG-nitro-L-arginine methyl ester, inhibitors of nitric oxide synthetase, did not appreciably inhibit the reaction. We conclude that stable, non-flavine-requiring oxidases, possibly cyclooxygenases or lipoxygenases, produced the H2O2 measured histochemically by our DAB reaction. These studies were made on tissue sections of acute dermal inflammatory lesions produced in rabbits by the topical application of 1% sulfur mustard [bis(2-chloroethyl) sulfide] in methylene chloride. Both intact PMNs and disintegrating PMNs in the base of the crust produced H2O2. Despite the production of H2O2 and the presence of peroxidase activity, no tissue damage was seen microscopically near the H2O2-producing cells, which indicates that the tissues are well protected by the antioxidants present in this self-limiting inflammatory reaction.

Antimicrobial therapy quickly eradicates susceptible bacilli, but the fight against drug-resistant disease must be waged entirely by host defenses. Knowledge of the two main types of immune response against tuberculosis and of how to manipulate those mechanisms--leading to precisely designed recombinant BCG vaccines--is essential to mounting an effective attack on the current epidemic.

Specific immediate and delayed (24-hour) local dermal reactivity to oxazolone and dinitrochlorobenzene (DNCB) was passively transferred to naive guinea pigs by the intradermal injection of immune guinea pig serum and its IgG1 fraction. In both actively sensitized and passively sensitized animals, neutrophils were the major cells present in the immediate reaction to the specific contactant. Basophils and mononuclear cells were the major cells present in the delayed reaction to the contactant. This late-phase reaction is, therefore, a cutaneous basophil hypersensitivity (CBH) response. The serum factor that passively transferred this CBH response was stable when heated at 56 degrees C for 1-4 h. Since transfer factor, cutaneous basophil hypersensitivity factor and guinea pig IgE are heat-sensitive, these factors probably made little or no contribution to this response. Because proteinase inhibitors are known to inhibit mast-cell degranulation in vitro, we tested the effect of soybean proteinase inhibitor in vivo. This inhibitor suppressed both the immediate and the delayed skin reactivities mediated by intradermal contactant-specific IgG1. These studies support the following concept: IgG1 in guinea pigs (and IgE in human beings) sensitize mast cells to specific antigens. Such antigens degranulate mast cells, releasing histamine and other mediators for the immediate hypersensitivity reaction, and cause mast cells to produce cytokines that recruit basophils, eosinophils and mononuclear cells for the late-phase (CBH) reaction.

Guinea pigs were sensitized by the topical application of either dinitrochlorobenzene (DNCB) or oxazolone on days 1, 2, 3, and 10. Seventeen days after the first treatment with the sensitizer, full-thickness 1.0-cm2 explants of untreated areas of skin were topically exposed in vitro to these contactants. Compared to the response of skin from control guinea pigs, skin from specifically sensitized animals showed a dose-related increase in the number of epidermal cells containing vacuoles. A specific increase in epidermal microblistering paralleled the increase in epidermal vacuolization. In addition, skin explants from sensitized animals (exposed to the contactant) showed a specific decrease in the incorporation of [14C]leucine. Full-thickness skin explants from unsensitized guinea pigs were sensitized in vitro by the intradermal injection of serum IgG1 fraction from oxazolone-sensitized guinea pigs. In such passively sensitized explants, the specific contactant produced an increase in the number of epidermal vacuoles, an increase in the amount of microblistering, and a decrease in the number of mast cells detectable by Giemsa staining. To elicit this specific response, the concentration of the specific contactant had to be mildly injurious, as well as antigenic. This requirement for nonspecific injury could be met by topically exposing skin explants to a nonspecific irritant followed by a sub-threshold concentration of the specific contactant. In contrast to vacuole formation and blistering, contactant-specific degranulation of mast cells (measured by the decrease in their number) did not require irritant levels of the contactant. These studies show that several components of contact sensitivity reactions can be reproduced in vitro by the passive transfer of sera containing antigen-specific immunoglobulins. Banks of such sera might, therefore, be useful in identifying (in human populations) many pre-existing sensitivities to chemical compounds.

It is widely believed that cell-mediated immunity and the associated ability of macrophages to destroy or inhibit the bacillus are all that is required to control pulmonary tuberculosis. However, although cell-mediated immunity is a major host defense against the tubercle bacillus, it is fully effective only in one of the four stages of the disease. Here, Arthur Dannenberg describes the entire pathogenesis of tuberculosis, with illustrations from the rabbit model of M.B. Lurie. In addition, he documents that the delayed-type hypersensitivity reaction (producing tissue necrosis) greatly benefits the host by arresting the logarithmic growth of bacilli within immature macrophages.

Peak (1 and 2 d) and healing (3, 6, and 10 d) inflammatory lesions were produced in rabbits by the topical application of the military vesicant, bis(2-chloroethyl)sulfide, commonly called sulfur mustard (SM). SM produces an acute sterile dermal inflammatory reaction with little or no necrosis, except in the epidermis, which dies during the first day. After an animal was killed, its lesions were excised intact, as full-thickness 1.0-cm2 explants. They were then organ-cultured for 3 d in order to maintain the viability of both local and infiltrating cells. The extracellular fluid in each lesion equilibrated with the culture fluid, which was collected daily and analyzed for collagenase and proteoglycanase activities. These metalloproteinase activities were measured after we had i) destroyed the alpha-macroglobulin inhibitors with KSCN, ii) destroyed the tissue inhibitor of metalloproteinases (TIMP) by reduction and alkylation, and iii) activated the latent proteinase activity with aminophenylmercuric acetate (APMA). Hydroxyproline-containing peptides and glycosaminoglycans (GAG) released into the culture fluids were also measured as indicators of local collagenase and proteoglycanase activity within the inflammatory lesions. In general, the levels of both the metalloproteinases and the products of their activity were higher in second- and third-day culture fluids than in first-day culture fluids, and higher in fluids from SM lesions than in those from normal skin. The activated fibroblast was apparently the major cell type producing the collagenase and proteoglycanase. The hydrolysis of collagen and ground substance occurs pericellularly. An excess of inhibitors exists outside the pericellular region. The daily change in culture fluids apparently decreased such inhibitors, so that by the second and third day of culture we could detect the changes in pericellular enzyme activity that were not detectable on the first day of culture. As the inflammatory lesions healed, the extracellular enzyme products (hydroxyproline and GAG) increased more than the enzymes that produced these products. With healing, a decrease occurs in the extravasation of all serum components, especially the large ones such as the alpha-macroglobulin inhibitors. We propose that during healing, the decrease in these inhibitors allows the metalloproteinases to begin the remodeling process, and that during the peak phase of inflammation, these same inhibitors protect extracellular matrix against hydrolysis by such proteinases.

This report describes a model organ-culture system for testing the toxicity of chemical substances that are topically applied to human skin. In this system, the viable keratinocytes in the full-thickness skin explants are protected by the same keratinized layer as skin remaining on the donor, and toxicity can be assessed microscopically and/or biochemically. The human skin specimens were discards from a variety of surgical procedures. They were cut into full-thickness 1.0-cm2 explants, and briefly exposed to the military vesicant sulfur mustard (SM), which was used as a model toxicant. The explants were then organ cultured in small Petri dishes for 24 h at 36 degrees C. In the 0.03-1.0% dosage range, a straight-line dose-response relationship occurred between the concentration of SM applied and the number of paranuclear vacuoles seen histologically in the epidermis. Within the same SM dosage range, there was also a proportional decrease in 14C-leucine incorporation by the explants. Thus, the number of paranuclear vacuoles reflected decreases in protein synthesis by the injured epidermal cells. The epidermis of full-thickness untreated (control) human skin explants usually remained viable for 7 d when stored at 4 degrees C in culture medium. During storage, a relatively small number of paranuclear vacuoles developed within the epidermis, but the explants were still quite satisfactory for testing SM toxicity. Incubation (for 4 or 24 h at 36 degrees C) of such control skin explants reduced (often by 50%) the small number of paranuclear vacuoles produced during 4-7 d of storage. This reduction was probably caused by autolysis of many of the vacuolated cells. Two types of paranuclear vacuoles could be identified by both light and electron microscopy: a storage type and a toxicant type. The storage type seemed to be caused by autolysis of cell components. The toxicant type seemed to be caused by an invagination of the plasma membrane. Only toxicant-type vacuoles increased appreciably in number when skin explants were exposed to mustard, and to other toxicants.

Explanted hepatic granulomas, eosinophils obtained from the peritoneal cavity of schistosome-infected mice, schistosome egg granuloma macrophages, alveolar macrophages, and activated peritoneal macrophages obtained from Listeria-infected mice were miracidicidal when cultured at 21% oxygen. This activity was markedly attenuated at physiologic oxygen concentrations (1-15%). Catalase and superoxide dismutase blocked the miracidicidal activity of inflammatory cells but did not prevent granuloma-mediated egg killing. However, the biomimetic superoxide dismutase, copper (II) [diisopropyl salicylate]2, inhibited granuloma-mediated egg killing in a dose-dependent, apparently nontoxic manner. Thioglycollate-elicited macrophages did not kill schistosome egg miracidia even when cultured in 21% oxygen, unless pretreated with lipopolysaccharide. Isolated schistosome eggs initiated an oxidative burst in macrophages, as measured by superoxide anion production. This burst was suppressed at reduced oxygen concentrations. Thus schistosome egg miracidia can be killed nonspecifically by macrophages through the release of cytotoxic reactive oxygen intermediates triggered by the egg. This activity is not supported by the oxygen concentrations found in most tissues, with the possible exception of the lung. Schistosoma mansoni eggs, injected intraveneously and lodged in the pulmonary vasculature of mice, were killed rapidly, with a half life of 3.5 days. Eggs, injected into the mesenteric veins and lodged in the liver, remained fully viable for several weeks. The data suggest that the high oxygen tension of the lung allows for the increased production of reactive oxygen intermediates (ROI) by local inflammatory cells, which in turn increases their miracidicidal efficiency. Conversely, the relatively hypoxic environment of the liver decreases ROI production by local inflammatory cells and decreases their miracidicidal efficiency.

Paired groups of male rabbits were challenged with Treponema pallidum and Mycobacterium bovis BCG. One group had been sensitized to BCG by inoculation 3 weeks before challenge. All animals were challenged intradermally at multiple sites with T. pallidum alone, BCG alone, and both organisms into the same sites. The resulting lesions were followed clinically and histologically. BCG lesions enlarged more rapidly in sensitized rabbits, but they were otherwise no different from those in the controls. T. pallidum lesions enlarged and regressed simultaneously in both groups, but in the BCG-sensitized animals they became twice as large as those in the unsensitized rabbits. Mixed BCG-T. pallidum lesions showed the greatest differences in the two groups of animals. Like the pure BCG lesions, they enlarged more rapidly in the sensitized rabbits but began to recede after 1 week. The corresponding lesions in the controls enlarged more slowly and reached their maximum size after 3 weeks when the receding lesions in the sensitized animals were much smaller. The most marked histological-histochemical difference between the two groups of animals was in the number and activation of macrophages. These cells were more numerous in the mixed lesions of BCG-sensitized animals than in similar lesions of the controls and more activated as determined by beta-galactosidase staining. Although sparsely distributed, activated macrophages were more numerous in the pur T. pallidum lesions of sensitized animals than in those of control animals. Silver-stained sections revealed fewer treponemes in mixed lesions of sensitized animals than in the mixed lesions of control animals. Quantitation of treponemes in pure T. pallidum versus mixed lesions was determined in two groups of rabbits challenged intratesticularly. The total number of treponemes per testis in the mixed lesions of BCG-sensitized rabbits was significantly less than the number in the mixed lesions of control animals, and also less than the number in pure T. pallidum lesions of both groups of animals.

Rabbits were injected intramuscularly with cortisone acetate (2 mg/kg) on alternate days. Six days after the first injection these rabbits and controls were injected intradermally in multiple sites with BCG (the vaccine strain of tubercle bacillus). Periodically, over the next 2 months, the resulting lesions were measured and surgically biopsied, and the animals were tuberculin-tested. Macrophage activation in the BCG lesions was evaluated histochemically by staining for beta-galactosidase activity. Both BCG lesions (and tuberculin reactions) in the cortisone-treated group were considerably smaller than those in the control group. Cortisone was highly effective in reducing the number of infiltrating mononuclear cells (MN), the amount of caseous necrosis and ulceration, and the percent of NM that were beta-galactosidase-positive. The decreased activation and reduced number of macrophages readily explains the increased susceptibility to tuberculosis found amoung patients receiving glucocorticosteroids. In the BCG lesions, the local decrease in the number and function of leukocytes probably explains the decreased tissue necrosis. Such antiinflammatory effects of corticosteroids may offset, in selected antimicrobial-treated cases, the hormone's detrimental effect on host resistance to infectious agents.

To evaluate extracellular hydrolytic enzymes in an in vivo system, plastic chambers were glued over rabbit dermal BCG lesions in various stages of development, after the central epithelium was removed with a scalpel. They were filled with tissue culture medium and left in place 2 days. The following enzymes in the fluid were assayed: collagenase (an enzyme secreted but not stored in macrophages); lysozyme (both secreted and stored); DNase and RNase (released on cell death and possibly regurgitated but not secreted); and, as a control, lactic dehydrogenase (released only on cell death). Tissue sections were prepared and studied histologically for the type of cell infiltrate, for beta-galactosidase (our marker enzyme for macrophage activation), and for necrosis. At 11 and 18 days of age the BCG lesions were largest and the number of activated macrophages in the chamber beds was highest. At this time the levels of the five enzymes assayed in the chamber fluids reached their peaks, tuberculin hypersensitivity was well developed, and the bacilli components would still be plentiful. In general, the chamber fluids from 11- and 18-day BCG lesions contained higher enzyme levels than chamber fluids from tuberculin reactions. Active collagenase was only detected in fluids from such BCG lesions. Evidently, the serum in the chamber fluids was sufficient to inhibit the lower amounts of collagenase probably released from smaller BCG lesions and tuberculin reactions (and from the 2-week polystyrene lesions that were also evaluated). These studies demonstrate that in chronic inflammatory reactions, both acid-acting and neutral-acting hydrolytic enzymes are released extracellularly. Tissue components would be hydrolyzed locally wherever the acid-acting hydrolytic enzymes encounter a drop in pH and wherever the concentration of neutral-acting hydrolytic enzymes exceeds the concentration of their inhibitors.

Acid-acting (pH 6-7) (presumably lysosomal) ribonuclease and neutral-acting (pH 7-8) calcium-dependent phospholipase A2 (presumably the enzyme releasing arachidonic acid from membrane phospholipids) were demonstrated by the peroxidase-antiperoxidase (PAP) immunocytochemical technique in rabbit professional phagocytes: pulmonary alveolar macrophages (AM), oil-induced peritoneal exudate macrophages (M phi) and glycogen-induced peritoneal exudate polymorphonuclear granulocytes (PMN). All three cell types stained positively with antisera to purified rabbit lung RNase and purified rabbit granulocyte phospholipase A2. The RNase and phospholipase A2 were also demonstrated by the PAP technique in the activated macrophages and granulocytes present in tissue sections of tuberculous (BCG) lesions. The intensity of staining of these two enzymes in individual macrophages did not change appreciably as the BCG lesions developed and regressed, but there were more macrophages rich in both enzymes when the lesions reached their peak size at 21 days. When the anti-RNase serum was fractionated by immunoabsorbent chromatography, the anti-delta RNase serum fraction stained exudate M phi and PMN better than AM; and the anti-beta RNase fraction stained AM better than M phi and PMN. Similar to isolated phagocytes, tissue granulocytes stained best with the anti-delta fraction; and activated tissue macrophages stained best with the anti-beta fraction. Thus, macrophages and granulocytes contain two types of RNase, beta and delta; and the beta RNase is associated with macrophage activation.

Developing and healing inflammatory lesions were topically produced in the skin of rabbits by sulfur mustard (SM). After the rabbits were sacrificed, the various lesions were removed and organ-cultured. The organ-culture fluids extracted the extracellular lysosomal enzymes (acid phosphatase, beta-glucuronidase, beta-galactosidase, and lysozyme), so that they could be measured biochemically along with lactic dehydrogenase (LDH), an enzyme marker for cell death. In tissue sections, the number and types of cells were counted, and their lysosomal enzyme content evaluated histochemically. The culture fluids from peak lesions contained much lower levels of all five enzymes than did culture fluids from healing lesions. When histological-histochemical-biochemical correlations were made, serum, macrophages (MN), and activated fibroblasts (but not tissue PMN) appeared to be major sources of extracellular lysosomal enzymes in peak lesions; and the dead PMN in the crusts and the activated fibroblasts in the tissues appeared to be major sources in healing lesions. The high lysosomal enzyme content of the crusts covering the lesions suggests that this passive barrier may also play an active role in promoting healing and in protecting against invasion by microorganisms.

A simple, cold-embedding method (on cracked ice at 2 C) is presented for the demonstration of acid phosphatase, beta-galactosidase and nonspecific esterase in glycol methacrylate tissue sections.

Cathepsin D was visualized in free pulmonary alveolar macrophages (AM), in oil-induced peritoneal macrophages (MN) and in rabbit pulmonary and dermal BCG lesions with unlabeled antibodies and the peroxidase-antiperoxidase (PAP) complex. Large amounts of cathepsin D were present in AM and lower amounts in MN. In the lung this enzyme was richest in the alveolar macrophages that accumulated around the BCG lesions. In the dermal lesions, cathepsin D was in highest concentration in macrophages at the border of the necrotic (liquefying) centers. It was also found in high concentration in keratinizing cells of the dermal epithelium and hair follicles. It did not, however, increase appreciably in many of the activated macrophages that stained intensely for the lysosomal enzyme beta-galactosidase. In fact, many epithelioid cells with high beta-galactosidase activity contained no visible cathepsin D. This proteinase does not, therefore, seem to be primarily involved in the lymphocyte-mediated macrophage activation associated with acquired cellular resistance to tubercle bacilli. It is probably more involved with cell autolysis, with the digestion of ingested necrotic debris and, in all likelihood, with the process of liquefaction, the most adverse event in the pathogenesis of tuberculosis in man.

Extravasated serum seems to be the major modulator of the local inflammatory response, because it provides both proinflammatory and antiinflammatory components. This report describes the rates of entry and turnover of extravasated serum protein in dermal inflammatory lesions produced by the military vesicant sulfur mustard (SM). Rabbits, bearing SM skin lesions, were given an intravenous injection of Evans blue dye, so that at the time of sacrifice, 2 hours later, their skin lesions were 2 hours and 1,2,3,6, and 10 days of age. Evans blue labels serum albumin, a representative serum protein. By multiplying the amount of Evans blue contained in the lesions by a factor that converted micrograms of Evans blue into milligrams of serum protein, the authors could estimate the 2-hour rate of entry of serum protein into these lesions. Serum protein in the lesions was both bound and unbound. The unbound protein was extractable from the lesions into the culture fluids, and, electrophoretically, was similar in composition to serum protein. Grossly edematous peak lesions (1 day of age) contained 7.8 mg of unbound serum protein per square centimeter of skin. Healing lesions (6 and 10 days of age) contained about 4.5 mg/sq cm, and normal skin about 1.7 mg/sq cm. Lesions 1 day of age had the highest rate of serum albumin entry, and about 36% of this Evans-blue-labeled protein was unbound, ie, extractable into the culture fluids. Lesions 3 and 6 days of age had a rate of serum albumin entry that was roughly half that of 1-day lesions, and only about 13% of this entering protein was unbound. Normal skin had a very low rate of serum albumin entry, and only 8% of this entering protein was unbound. The turnover rate of the unbound (extractable) serum protein could be estimated from the 2-hour entry rate of the Evans-blue-labeled albumin and the total protein in the culture fluids. In 1-day lesions, about 25% of the serum protein in the culture fluids was protein which had entered during the last 2 hours, so that 100% of this unbound protein should have been replaced once in 8 hours. In contrast, in 3- and 6-day lesions, this unbound serum protein should have been replaced once in about 35 hours, and in normal skin once in 80 hours. Evans-blue-labeled serum albumin continuously entered both the bound and unbound compartments of the SM lesions, even during the healing stages.(ABSTRACT TRUNCATED AT 400 WORDS)

The acid-acting proteinase, cathepsin D (EC 3.4.4.23), was purified from extracts of homogenized rabbit lung and beef lung by autolysis at acid pH, acetone and ammonium sulfate fractionation, column chromatography, and isoelectric focusing. Four isoenzymes were obtained from each source. With acid hemoglobin as the substrate, the proteinase from rabbit lung had a pH optimum of 3.0 and that from beef lung had a pH optimum of 3.6. Their activity was not affected by thiol reagents or by Fe(2+), Mn(2+), or Mg(2+). One isoenzyme from rabbit lung was used to immunize a goat, and one from beef lung was used to immunize a rabbit. In immunoelectrophoresis, each resulting antiserum formed a single precipitin line with its homologous enzyme. They cross-reacted with the other three isoenzymes from the same species, but not with any isoenzyme from the other species. At high concentrations, each antiserum completely inhibited the proteolytic activity of its homologous enzyme. The antiserum against rabbit lung cathepsin D also inhibited the proteolytic activity of rabbit peritoneal and pulmonary macrophages. In limited quantities, this antiserum has now been made commercially available and is being used with labeled antibody techniques to identify under a microscope the presence of cathepsin D in macrophages and to study its role in the pathogenesis of tuberculosis.

This is the third report in a series on the inflammatory mediators and modulators released in organ culture from skin lesions of various ages, which were produced in vivo in rabbits by the military vesicant, sulfur mustard (SM). It describes the electrophoretic protein fractions and trypsin-inhibitory capacities of the various culture fluids and the amounts of alpha 1-proteinase inhibitor and alpha-macroglobulin proteinase inhibitors in these fluids. With one-dimensional electrophoresis, the albumin and beta-globulin fractions of protein in culture fluids varied little with the development and healing of the SM lesions. These fractions proportionally resembled the corresponding fractions found in serum. The alpha 1-globulin fraction was proportionally smaller than the corresponding fractions of serum as the lesions healed. The alpha 2-globulin fraction was proportionally smaller than the corresponding fractions of serum at all stages of lesion development and healing. The gamma-globulin fraction was proportionally larger as the lesions healed. With two-dimensional electrophoresis, about 68%, 46%, and 35% of the protein spots in culture fluids from representative 1-day and 6-day SM lesions and normal skin, respectively, matched those from serum. In each case, the large, diffuse, serum albumin spot represented about two-thirds of the protein present. Thus, gravimetrically, in normal skin and in both developing and healing lesions, the extracellular proteins were 80-90% of serum origin. The trypsin-inhibitory capacity (TIC) per milligram protein in the culture fluids of healing lesions was markedly less than the TIC per milligram protein in the fluids of peak lesions. This decrease correlates well with the decrease found in the alpha 1-globulin fraction, which contains alpha 1-antiproteinase (alpha 1-PI) (and alpha 1-macroglobulin [alpha 1M] in rabbits). The alpha 1PI and the alpha 1M-alpha 2M proteinase inhibitors were identified in the culture fluids by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blots, specific antibodies, and the immuno-peroxidase technique. The levels of both free and proteinase-complexed alpha 1PI and alpha M inhibitors in the culture fluids decreased as the lesions healed. In both developing and healing lesions, at least half of the alpha 1PI and alpha M inhibitors seemed to be complexed with proteinases. Thus, serum seems to be a major source of unbounded extracellular protein within acute inflammatory lesions, and serum proteinase inhibitors seem to be the host's major defense against local damage by proteinases from serum, infiltrating leukocytes, and activated fibroblasts.

We studied the effects of repeated exposures of antigen on airway reactivity to mediators of anaphylaxis and immediate response to the antigen. Seven antigen-sensitive sheep were exposed to aerosols of Ascaris suum antigen 5 times biweekly; a control group of seven sheep underwent the same exposure regimen with saline vehicle. Sheep were assigned to experimental (Ascaris) or control groups so the distribution of animals with regard to bronchial reactivity to mediators was about the same. Airway reactivity was assessed by determining the effects of aerosolized histamine (10-1,000 micrograms), prostaglandin F2 alpha (PGF2 alpha, 10-300 micrograms), and a stable analogue of thromboxane A2 (U-46619, 1-100 micrograms) on lung resistance (RL) and dynamic lung compliance (Cdyn). Before treatment, experimental and control groups showed similar changes in RL and Cdyn, with analogue greater than histamine greater than PGF2 alpha. At the highest dose of each agonist, mean increases in RL were 50, 123, and 29%, respectively, and mean decreases in Cdyn were 21, 45, and 12%. During the first 15-min exposures to antigen aerosol, mean RL had increased by 125% and Cdyn decreased by 38% of base-line values; hyperinflation following the exposures reduced the changes to 56 and 31%, respectively. Changes in RL and Cdyn during the final antigen exposures and following postexposure hyperinflation were reduced significantly (P less than 0.05) compared with the initial exposures. Baseline RL and Cdyn before and after the exposures to antigen or saline were not significantly different. Airway reactivity to histamine, PGF2 alpha, or analogue was not significantly altered in these atropinized animals over the range of doses studied.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of these studies was to identify some of the extracellular proteolytic enzymes associated with the development and healing of acute inflammatory lesions. Lesions were produced in the skin of rabbits by the topical application of the military vesicant, sulfur mustard (SM). Full-thickness, 1-cm2 central biopsies of the lesions were organ-cultured for one to three days, and the culture fluids were assayed for proteases with a variety of substrates. When compared to culture fluids from normal skin, the culture fluids from both developing and healing SM lesions had three to six times the levels of proteases hydrolyzing two synthetic peptide substrates: (1) t-butyloxycarbonyl-Leu-Gly-Arg-4-trifluoromethylcoumarin-7-amide(Boc-Leu -Gly- Arg-AFC, herein abbreviated LGA-AFC), and (2) N-benzoyl-phenylalanine-beta-naphthyl ester (BPN). LGA-AFC is a substrate for trypsin, plasmin, plasminogen activator, thrombin, kallikrein, and the C3 and C5 convertases; BPN is a chymotrypsin and cathepsin G substrate. The culture fluids did not consistently hydrolyze four other synthetic peptide substrates or the proteins [14C]-casein and [14C]elastin. In order to determine the likely sources of LGA-AFCase and BPNase activity, we counted the number of granulocytes (PMNs), macrophages (MNs) and activated fibroblasts in histologic sections of developing and healing SM lesions, and we measured the levels of these enzymes in serum, in culture fluids of PMN and MN peritoneal exudate cells, and in culture fluids of two fibroblast cell lines. In SM lesions, serum and fibroblasts seemed to be the major source of LGA-AFCase, and serum alone the major source of BPNase. Tissue PMNs and MNs seemed to be only minor sources. The crusts of healing lesions, which were full of dead PMNs, seemed to be a rich source of both enzymes. In the SM lesion culture fluids, whether LGA-AFC and BPN were hydrolyzed by endopeptidases or only by exopeptidases could be determined by evaluating complex formation with alpha-macroglobulin proteinase inhibitors (alpha M). Endopeptidases, but not exopeptidases, are entrapped and inhibited by alpha M, because an internal peptide band in alpha M must first be hydrolyzed before molecular rearrangement (required for proteinase inhibition) occurs. The catalytic site of endopeptidases that are entrapped and inhibited by alpha M is known to remain active on (and reachable by) small synthetic peptide substrates such as LGA-AFC and BPN.(ABSTRACT TRUNCATED AT 400 WORDS)

BCG lesions were produced in the skin of rabbits, and biopsies were performed at 7, 21, and 42 days, when they were developing, maximal in size, and almost healed, respectively. Tissue sections were prepared and stained histochemically for several enzymes. The percentage of cells stained for a given enzyme and the distribution of such cells within lesions of various ages were determined. Seven-day BCG lesions contained few esterase- and beta-galactosidase-positive macrophages, but 21-day lesions contained many, especially in the viable and nonviable tuberculous granulation tissue at the edge of the now prominent caseous necrotic center. Both 7-day and 21-day lesions contained many acid phosphatase- and cathepsin-D-positive macrophages, which were numerous in the more peripheral parts of the lesion, where little or no necrosis was present. Enzyme patterns in 42-day lesions resembled those in 21-day lesions. The role of each of these enzymes in the development and regression of the BCG lesion is unknown. Nonetheless, these studies clearly demonstrate that this macrophage population is heterogeneous and that macrophages carry out different functions in different parts of the lesion at different times. Histochemical techniques were developed to stain two enzymes in the same tissue section. The first stain usually contained a naphthol substrate and produced a red color; the second stain contained an indoxyl substrate and produced a blue color. A cell staining with both was colored purple. The peroxidase-antiperoxidase immunocytochemical technique for cathepsin D (producing a red color) was also employed. 1) Red esterase (hydrolyzing naphthol AS-D acetate) and beta-galactosidase, and 2) red esterase and blue esterase (hydrolyzing 5-bromo-4-chloro-indoxyl acetate), probably the same enzyme, were usually present in the same macrophage. In contrast, each of the following enzyme pairs was usually present in a different macrophage: 3) cathepsin D and beta-galactosidase, 4) cathepsin D and blue esterase, 5) acid phosphatase and beta-galactosidase, and 6) acid phosphatase and blue esterase. Roughly 10% of the macrophages stained for one enzyme existed side by side with macrophages stained for a different enzyme. These results suggest that local macrophage activation is under two levels of control. The first, macrolocal control, would determine the overall enzyme distribution in the lesion; whereas the second, microlocal control, would determine enzyme distribution on a cell-by-cell basis, ie, how two neighboring macrophages can each be rich in a different enzyme.

Chickens were exposed to SO2 in relatively low concentrations (3.4 to 18.5 parts per million (ppm)) for 1 to 14 days. A portion of their tracheas was embedded in water-soluble methacrylate, cut at 2 micrometer and stained with hematoxylin and eosin, Wright's stain, methyl green-pyronin, Alcian blue - periodic and Schiff, and for acid phosphatase. An increase was found in (a) the mucosa to wall ratio; (b) the number of mucosal cells in mitosis; (c) the number of macrophages, lymphocytes, plasma cells, and neutrophils in the epithelium and lamina propria; and (d) the number of these infiltrating cells which contained acid phosphatase. The number of mucus- and seromucus- secreting cells and vasoamine-containing cells were sometimes increased, but not consistently. The percentage of cells containing sialidase-sensitive sialomucins was elevated, and percentage of cells containing neutral mucins was reduced. These changes were only partly related to the SO2 concentration and the duration of SO2 exposure, in that increasing amounts of SO2 did not always cause increasing changes in the mucin composition. Evidently, the altered mucins sometimes protected against further mucin modification.

Sulfur mustard (SM) (di-2-chlorethyl sulfide), used for chemical warfare in World War I, is a highly reactive radiomimetic alkylating agent. When applied to the skin of rabbits and guinea pigs, it produced vascular leakage, leukocyte infiltration, and slow death of basal epidermal cells. Thirty to sixty minutes after exposure to SM, injury to the superficial microvasculature (beneath the SM application site) was detected by measuring vascular leakage with Evans blue dye and also with horseradish peroxidase. At this same time, injury to the superficial fibroblasts was observed ultrastructurally; and an unexpectedly high percentage of basophils was found among the early infiltrating granulocytes. At 2 to 4 hr, the vascular leakage ceased, and had resumed by 8 hr in a more diffuse form. At this time, the basal epidermal cells showed pyknotic nuclei, an increase in their lysosomal enzymes (observed histochemically), and autophagic vacuoles (observed ultrastructurally). Leukocyte infiltration was marked, consisting mostly of heterophils (PMN) with a reduced percentage of basophils. During the next 24 to 72 hr, the entire inflammatory reaction reached its peak; and a superficial, crust-covered ulcer developed. Then, over the next 10 days, the lesion gradually subsided with concomitant repair and healing. Glucocorticosteroids decreased the early edematous phase, but did not affect the rate of healing. These findings suggest that the skin response to sulfur mustard has an immediate and a delayed phase. The immediate phase, i.e., within the first hour, was characterized by injury to the superficial fibroblasts and to the endothelium of superficial capillaries and venules, possibly because of direct damage to their cell membranes. At this time, a restricted vascular leakage and a selective granulocyte infiltration containing many basophils occurred. The delayed phase, which became evident after 8 hr, was characterized by the death of basal epidermal cells, probably because of DNA damage. This phase was accompanied by generalized vascular leakage, by massive heterophil immigration, and eventually by ulceration.