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The variable efficacy of bacillus Calmette-Guérin (Mycobacterium bovis BCG) in protecting humans and cattle against tuberculosis has prompted a search for a more effective vaccination regimen. A prime-boost strategy was investigated in cattle naturally sensitized to environmental mycobacteria by using a combination of three DNA vaccines coding for Hsp 65, Hsp 70, and Apa for priming, followed by a boost with BCG prior to experimental challenge with virulent M. bovis. Controls were vaccinated with DNA or BCG alone or were not vaccinated. The immune responses were monitored throughout the study, and protection was assessed based on reductions in the numbers of lesions and viable mycobacteria in lymph node samples. Vaccination with BCG alone or with a DNA prime-BCG boost regimen induced high levels of antigen-specific gamma interferon (IFN-gamma) in whole-blood cultures. In the prime-boost group there were fewer animals with severe lung lesions, fewer lymph nodes with lesions per animal, a smaller proportion of animals with lesions, lower mean lung and lymph node lesion scores, and less M. bovis isolated from retropharyngeal and thoracic lymph nodes compared to the results obtained for the nonvaccinated animals. The prime-boost regimen induced significant enhancement of protection in six parameters, compared with significant enhancement of protection in only two parameters for BCG alone. In addition, following challenge, in vitro IFN-gamma responses against ESAT-6 and CFP-10, as well as bovine tuberculin-induced skin test and in vitro IFN-gamma responses, were identified as immunological markers that predicted protection. The use of the prime-boost strategy suggested that a combination of vaccines may be better than a single vaccine for protection against tuberculosis.

Tuberculous pleurisy is a severe inflammatory response induced by Mycobacterium tuberculosis organisms that have escaped from lung granulomata into the pleural space during pulmonary infection. We have used the guinea pig model of tuberculous pleurisy to examine several aspects of the immune response to this antigen-specific inflammatory event. Pleurisy was induced by injection of heat-killed M. tuberculosis H37Rv directly into the pleural space of guinea pigs previously vaccinated with M. bovis BCG. Four animals were euthanized each day over a period of 9 days. Fluid in the pleural cavity was analyzed for transforming growth factor beta 1 (TGF-beta 1) and total interferon (IFN) protein levels. In addition, RNA was obtained from pleural cells and examined for TGF-beta 1, tumor necrosis factor alpha (TNF-alpha), IFN-gamma, and interleukin-8 (IL-8) expression by real-time PCR. Finally, pleural cells were examined for the ability to proliferate in response to concanavalin A and purified protein derivative (PPD) in vitro. In the pleural fluid, TGF-beta 1 protein concentrations increased over the course of the inflammatory response while IFN protein levels were not significantly altered. Expression of TGF-beta 1 mRNA peaked on days 3 and 4, and IFN-gamma mRNA expression peaked on day 3 and then returned to background levels. TNF-alpha mRNA expression was highest on days 2 to 4, and IL-8 mRNA levels remained elevated between days 2 and 5, peaking on day 3 before returning to background levels. PPD-induced proliferative responses were evident by day 3 and remained present throughout the study. Analysis of cytokine expression during tuberculous pleurisy may lead to a better understanding of the self-healing nature of this manifestation of tuberculosis.

Purified protein derivative (PPD) RT23-recalled T-cell receptor (TCR) V beta expression was studied in the peripheral blood of 42 pulmonary tuberculosis patients and 44 healthy controls from southern India, a region where tuberculosis is endemic. Forty-eight-hour whole-blood cultures in the presence or absence of PPD-RT23 were set up, and at the end of the culture period total RNA was extracted and cDNA was synthesized. Expression of various TCR V beta families was assessed by using family-specific primers. PPD-specific expression (usage) of TCR V beta families 4, 6, 8 to 12, and 14 was found in more controls than patients. Among the responders (individuals who showed PPD-specific expression), endemic controls had significantly higher responses than the patients had for TCR V beta families 2, 3, 7, 13, and 17. The majority of the patients did not show usage of most of the TCR V beta families, and this was attributed to T-cell downregulation. A four-way nested classification analysis revealed that TCR V beta family 1, 5, 9, 12, and 13 usage in the context of HLA class II high-risk alleles (DRB1*1501, DRB1*08, and DQB1*0601) and Mycobacterium bovis BCG scar status were the determining factors in susceptibility and resistance to tuberculosis. The healthier status of controls was attributed to the wider usage of many TCR V beta families readily recalled by PPD, while the disease status of the patients was attributed to TCR V beta downregulation and the resultant T-cell (memory cell?) unresponsiveness. Host genetics (HLA status) and BCG vaccination (scar status) seem to play important roles in skewing the immune response in adult susceptibility to pulmonary tuberculosis through TCR V beta usage.

Mycobacterium bovis infection of cattle represents a natural host-pathogen interaction and, in addition to its economic and zoonotic impact, represents a model for human tuberculosis. Extravasation and trafficking of activated lymphocytes to inflammatory sites is modulated by differential expression of multiple surface adhesion molecules. However, effects of M. bovis infection on adhesion molecule expression have not been characterized. To determine these changes, peripheral blood mononuclear cells from M. bovis-infected cattle were stimulated with M. bovis purified protein derivative (PPD) or pokeweed mitogen (PWM) and evaluated concurrently for proliferation and activation marker expression. Stimulation with PPD or PWM increased CD25 and CD44 mean fluorescence intensity (MFI) and decreased CD62L MFI on CD4(+) cells from infected animals. CD62L MFI on PPD- and PWM-stimulated gammadelta T-cell receptor-positive (TCR(+)) and CD8(+) cells was also reduced compared to that of nonstimulated gammadelta TCR(+) and CD8(+) cells. Using a flow cytometry-based proliferation assay, it was determined that proliferating cells, regardless of lymphocyte subset, exhibited increased expression of CD25 and CD44 and decreased expression of CD62L compared to cells that had not proliferated. In contrast to proliferation, activation-induced apoptosis of CD4(+) cells resulted in a significant down regulation of CD44 expression. Lymphocytes obtained from lungs of M. bovis-infected cattle also had reduced expression of CD44 compared to lymphocytes from lungs of noninfected cattle. These alterations in surface molecule expression upon activation likely impact trafficking to sites of inflammation and the functional capacity of these cells within tuberculous granulomas.

Bovine paratuberculosis is caused by the infection of young calves with Mycobacterium avium subsp. paratuberculosis, resulting in a chronic granulomatous infection of predominantly the ileum. After an incubation period of 2 to 5 years, the disease becomes progressive in some of the chronically infected, but asymptomatic cows. This results in a protein-losing enteropathy that will ultimately be fatal. A loss of cell-mediated immune responses in symptomatic animals has been described, but no information is available concerning immune reactivity in the intestine. We sought to investigate putative disease status-associated lymphocyte subset distributions and antigen-specific functional characteristics of mononuclear cells isolated from blood, gut-associated lymphoid tissue, and the intestinal walls of 22 cows in different stages of disease and in control animals. The results demonstrated a significant decrease in CD4(+) T-cell frequency and a significant increase in TcR1-N12(+) gamma delta T-cell frequency in ileum lamina propria lymphocytes of symptomatic animals compared to the asymptomatic shedders. Immunohistology revealed that there was also an absolute decrease in the number of CD4(+) T cells in sections of the lesional ileum. Our findings also indicated that both peripheral and intestinal cell-mediated responses are decreased in symptomatic animals compared to asymptomatic animals. We conclude that the decrease in cell-mediated responses is likely related to a loss of antigen-specific CD4(+) T cells, which is most prominent in the lesional ileum from symptomatic animals, thus contributing to the progressive nature of bovine paratuberculosis.

The effect of Mycobacterium bovis BCG vaccination on interleukin-1 beta (IL-1 beta) or regulated-upon-activation, normally T-cell-expressed and -secreted chemokine (RANTES) mRNA expression in guinea pig spleen cells stimulated with concanavalin A, lipopolysaccharide (LPS), phorbol myristate acetate (PMA) plus ionomycin, or purified protein derivative (PPD) was studied in vitro. Similarly, peritoneal exudate cell-derived macrophages from naïve and BCG-vaccinated guinea pigs were infected with M. bovis BCG, Mycobacterium avium, the attenuated Mycobacterium tuberculosis H37Ra strain, or virulent strains H37Rv and Erdman of M. tuberculosis. Total RNA was subjected to Northern blot analysis using probes generated from guinea pig IL-1 beta or RANTES cDNA. Although IL-1 beta and RANTES mRNA could be detected in the spleen cells from naïve animals stimulated with LPS or PMA plus ionomycin, the levels were significantly enhanced after BCG vaccination. mRNA expression was also elevated in macrophages infected with live mycobacteria after BCG vaccination. However, macrophages infected with the virulent H37Rv strain of M. tuberculosis showed 75 to 90% reductions in IL-1 beta expression and 25 to 60% reductions in RANTES mRNA expression compared with macrophages infected with the attenuated H37Ra strain. The IL-1 beta mRNA levels peaked as soon as 1 h after PPD stimulation and 4 h after M. tuberculosis H37Rv infection of macrophages. In contrast, RANTES mRNA expression was delayed until 48 h after infection. These results indicate that molecular mediators produced in response to various stimuli associated with protective immunity against mycobacteria are upregulated after BCG vaccination; however, a significantly weaker response was observed with virulent M. tuberculosis. These initial studies indicate that BCG vaccination has a positive effect on IL-1 beta and RANTES mRNA expression by host cells in a highly relevant animal tuberculosis model.

Protein malnutrition leads to multiple detrimental alterations of host immune responses to mycobacterial infection. In this study, we demonstrated that splenocytes from low-protein (LP) guinea pigs vaccinated 6 weeks previously with attenuated Mycobacterium tuberculosis H37Ra failed to control the accumulation of virulent M. tuberculosis H37Rv in cocultured autologous peritoneal macrophages, despite the fact that they were able to control the accumulation of virulent tubercle bacilli in cocultured syngeneic peritoneal macrophages from normally nourished guinea pigs as successfully as did those from high-protein (HP) counterparts. Vaccine-induced growth control of virulent M. tuberculosis H37Rv in these cocultures appeared to be mediated by CD4 lymphocytes but not CD8 cells. Tuberculin (purified protein derivative [PPD])-induced lymphoproliferation was markedly impaired in vaccinated LP guinea pigs, and the depletion of CD4 lymphocytes significantly decreased lymphocyte proliferation whereas CD8 cell depletion did not. Protein malnutrition also impaired the abilities of cells from vaccinated LP guinea pigs to produce cytokines, including interferon, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta), in response to PPD, despite the demonstration of higher serum levels of TNF-alpha and TGF-beta after an intravenous injection of PPD into LP guinea pigs. In contrast, peritoneal macrophages from protein-malnourished guinea pigs produced a higher level of TGF-beta 4 days after infection in vitro with M. tuberculosis H37Rv than did those from protein adequate controls. These results suggest that dietary protein malnutrition impairs vaccine-induced resistance to M. tuberculosis, in part, by altering the cytokine profile to favor macrophage deactivation.

The induction of macrophage-deactivating (interleukin-10 [IL-10] and transforming growth factor beta [TGF-beta] and macrophage-activating (IL-1, IL-6, and tumor necrosis factor alpha [TNF-alpha] cytokines by lipoarabinomannan (LAM) from pathogenic Mycobacterium tuberculosis Erdman and H37Rv strains (ManLAM) and nonpathogenic mycobacteria (AraLAM) in human blood monocytes was examined. ManLAM was significantly less potent in induction of TNF-alpha, IL-1, IL-6, and IL-10 protein and mRNA, whereas its ability to induce TGF-beta was similar to that of AraLAM. Differences in induction of TNF-alpha mRNA by the two LAM preparations only became apparent at late time points of culture (24 h). The induction of TNF-alpha and IL-1 by purified protein derivative of M. tuberculosis was significantly stronger than that by ManLAM. Pretreatment of monocytes with ManLAM did not, however, interfere with cytokine induction by lipopolysaccharide or AraLAM. The extensive mannosyl capping of arabinose termini of ManLAM may underlie the lack of ability to induce some cytokines (IL-1, TNF-alpha, and IL-10) and the retained ability to induce TGF-beta. The latter may have a role in shifting the cytokine milieu in favor of survival of M. tuberculosis.

We examined the ability of purified protein derivative (PPD) of Mycobacterium tuberculosis to induce transforming growth factor beta 1 (TGF-beta 1), a potent immunosuppressive and macrophage-deactivating molecule, in blood monocytes from healthy individuals. TBF-beta 1 activity in PPD-induced monocyte supernatants was identified by Western immunoblot analysis and was not inhibited by polymyxin B, an inhibitor of bacterial lipopolysaccharide (LPS). Furthermore, PPD at equivalent amounts in weight to LPS was as potent in stimulation of monocyte production of TGF-beta 1 at 24 h of culture, as quantified by enzyme-linked immunosorbent assay. The inducing effect of PPD, in contrast to that of LPS, was sustained at later time points of culture (72 h). PPD enhanced the constitutive expression of TGF-beta 1 steady-state mRNA in monocytes at 24 and 48 h of culture. In contrast, neither mycobacterial heat shock protein (64-kDa protein of M.bovis) nor LPS induced TGF-beta 1 mRNA. Decay studies suggested a transcriptional rather than a posttranscriptional effect of PPD on TGF-beta 1 gene expression.

The expression of cytokine genes in cultures of human peripheral blood mononuclear cells (PBMC) stimulated with mannoprotein constituents (MP) of Candida albicans has been studied by means of S1 nuclease mapping analysis, polymerase chain reaction, and enzyme-linked immunosorbent assay. MP induced early, consistent, and long-lasting production of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha, and IL-6 mRNAs. Similar results were obtained when the same PBMC cultures were stimulated with the purified protein derivative (PPD) from Mycobacterium tuberculosis or with IL-2, although lower levels of IL-6 mRNA were detected in IL-2-stimulated cells than in MP- or PPD-stimulated cells. MP, PPD, and IL-2 induced appreciable levels of granulocyte-macrophage colony-stimulating factor and gamma interferon, but only MP and PPD were able to induce IL-2 mRNA. MP were unable to stimulate a consistent expression of the genes encoding for IL-4, IL-5, and IL-10, while low, sometimes barely detectable levels of these cytokine mRNAs were observed in PPD- or IL-2-stimulated PBMC cultures. When protein synthesis of MP-stimulated PBMC was inhibited by cycloheximide, a superinduction of mRNAs for IL-4 and IL-10 and, more markedly, gamma interferon was observed. Overall, these results highlight the powerful, selective induction of cytokine gene expression by MP constituents of C. albicans in human PBMC cultures, thus providing some functional clues to explain the efficient state of the anticandidal response in normal human subjects.

The ability to induce tuberculous pleuritis in Mycobacterium bovis BCG-vaccinated guinea pigs was investigated as a model of human disease. A pleural effusion of 5 to 10 ml was obtained 6 to 7 days after the bilateral pleural injection of a suspension of heat-killed M. tuberculosis cells. Histological lesions were indicative of granulomatous pleuritis. Comparative studies of T lymphocytes obtained from pleural fluid and peripheral blood revealed increased antigen-driven lymphoproliferation and E rosette formation in pleural effusion lymphocytes. The CD2+ T-lymphocyte population appeared to be expanded or concentrated in pleural fluid, suggesting a compartmentalization of antigen-reactive T lymphocytes. These data demonstrate that experimental tuberculous pleuritis with effusion, closely resembling the human disease, can be produced in BCG-vaccinated guinea pigs.

In this study the mitogenic and immunomodulating effects of bacterial cell wall preparations were investigated. Cell walls, peptidoglycans, and teichoic acids from Bacillus subtilis and Staphylococcus aureus Wood 45 activated both human T cells (supplemented with 10% monocytes) and B cells to proliferate and to produce leukocyte-inhibitory factor. Similar results were obtained with adult and umbilical cord blood cells, suggesting that these bacterial preparations were acting as mitogens. Cell walls and peptidoglycans had a modulating effect on purified protein derivative- or protein A-induced proliferation. In the presence of suboptimal concentrations of these stimulants, bacterial components enhanced the proliferative response. However, at optimal concentrations of purified protein derivative or protein A, bacterial components suppressed lymphocyte proliferation. Peptidoglycans solubilized by lysozyme activated B lymphocytes but not T cells. Solubilization had no effect on the immunomodulating capacity.

Concanavalin A (Con A) and phytohemagglutinin (PHA) released histamine from hamster mast cells incubated in a serum-free medium. Concentrations of Con A and PHA approximating those optimal for transforming lymphocytes also released maximal amounts of histamine without apparent cytotoxicity. Higher concentrations of mitogen inhibited both lymphocyte transformation and histamine release. Incubation at 37 C for 15 min released histamine, although longer times were more effective. Supernatants from cultured hamster splenocytes stimulated with Con A also released histamine from added mast cells. However, the effect could be inhibited by the addition of 0.1 M methyl alpha-D-mannoside or by passing the spleen cell culture supernatants through Sephadex G-75 to remove Con A. This mitogen-induced release of mast cell histamine is therefore not mediated by a lymphokine but probably results from a direct interaction of mitogens with receptors on mast cells.

Lymphocytes from uninfected, nonimmune mice can be stimulated to proliferate and produce antibody to diverse antigens in culture when exposed to the purified protein derivative of tuberculin or unheated tuberculin culture filtrate. Lymphocytes from numerous inbred strains of mice respond to tuberculin, but no true low responder strain has been found as yet. The evidence presented suggests that a subpopulation of B cells, which are distinct from those activated by lipopolysaccharide endotoxin, respond to tuberculin. The results of digestion experiments with insolubilized enzymes suggest that the nonspecific activating principles in tuberculin are protein.