A longitudinal characterization of the Non-Cystic Fibrosis Bronchiectasis airway microbiome.

A diverse microbiota exists within the airways of individuals with non-cystic fibrosis bronchiectasis (nCFB). How the lung microbiome evolves over time, and whether changes within the microbiome correlate with future disease progression is not yet known. We assessed the microbial community structure of 133 serial sputa and subsequent disease course of 29 nCFB patients collected over a span of 4-16 years using 16S rRNA paired-end sequencing. Interestingly, no significant shifts in the microbial community of individuals were observed during extended follow-up suggesting the microbiome remains relatively stable over prolonged periods. Samples that were Pseudomonas aeruginosa culture positive displayed markedly different microbial community structures compared to those that were positive for Haemophilus influenzae. Importantly, patients with sputum of lower microbial community diversity were more likely to experience subsequent lung function decline as defined by annual change in ≥-1 FEV1% predicted. Shannon diversity values <1 were more prevalent in patients with FEV1 decline (P = 0.002). However, the relative abundance of particular core microbiota constituents did not associate with risk of decline. Here we present data confirming that the microbiome of nCFB individuals is generally stable, and that microbiome-based measurements may have a prognostic role as biomarkers for nCFB.

LTB4 is present in exudative pleural effusions and contributes actively to neutrophil recruitment in the inflamed pleural space.

The pleural space is a virtual compartment between the lung and chest wall that becomes filled with fluid and inflammatory cells during a variety of respiratory diseases. Here, we study the potential role of the eicosanoid metabolite leukotriene B4 (LTB4) in disparate diseases leading to acute (pneumonia) or chronic (tuberculosis, cancer) inflammation of the pleural space. LTB4 concentrations were significantly higher in pleural fluid due to pneumonia, tuberculosis and cancer with respect to congestive heart failure and correlated with neutrophil elastase, which is used as an indication of state of activation of neutrophils in the pleural space. Moreover, pleural LTB4 was biologically active, as an anti-LTB4 antibody partially neutralized the chemotactic activity of parapneumonic, tuberculous and cancer effusions. Macrophages, neutrophils, lymphocytes, mesothelial cells and cancer cells all expressed mRNA for 5-lipoxygenase, the enzyme that initiates leukotriene synthesis leading to the production of LTB4, in exudative pleural effusions. Upon stimulation in transudative pleural effusions, pleural macrophages produced, in a time-dependent fashion, a significantly higher concentration of LTB4 than mesothelial cells. These studies demonstrate that different cell types are capable of producing LTB4 in the inflamed pleural space and that this mediator may play a crucial role in the recruitment of neutrophils into the pleural space.

Lipopolysaccharide-stimulated or granulocyte-macrophage colony-stimulating factor-stimulated monocytes rapidly express biologically active IL-15 on their cell surface independent of new protein synthesis.

Although IL-15 shares many of the biological activities of IL-2, IL-2 expression is primarily under transcriptional regulation, while the mechanisms involved in the regulation of IL-15 are complex and not completely understood. In the current study, we found that CD14(+) monocytes constitutively exhibit both IL-15 mRNA and protein. IL-15 protein was found stored intracellularly and stimulation of CD14(+) monocytes with either LPS or GM-CSF resulted in mobilization of IL-15 stores to the plasma membrane. This rapidly induced surface expression was the result of a translocation of preformed stores, confirming that posttranslational regulatory stages limit IL-15, because it was not accompanied by an increase in IL-15 mRNA and occurred independent of de novo protein synthesis. After fixation, activated monocytes, but not resting monocytes, were found to support T cell proliferation, and this effect was abrogated by the addition of an IL-15-neutralizing Ab. The presence of preformed IL-15 stores and the ability of stimulated monocytes to mobilize these stores to their surface in an active form is a novel mechanism of regulation for IL-15.

Phagocytosis and protein processing are required for presentation of Cryptococcus neoformans mitogen to T lymphocytes.

In addition to eliciting antigen specific T-cell-mediated immunity, Cryptococcus neoformans possesses a mitogen (CnM) that activates naive T cells to proliferate. This mechanism of T-cell activation is accessory cell dependent and major histocompatibility complex unrestricted. CnM-induced T-cell proliferation correlates with internalization of the organism, suggesting that intracellular processing is required to liberate CnM prior to presentation to T cells. To determine whether phagocytosis and processing are required, various inhibitors of accessory cell uptake and processing were used. C. neoformans was observed within the accessory cells. Paraformaldehyde fixation of the accessory cell abrogated presentation of CnM to T cells, indicating that a dynamic accessory cell surface was required. A lysosomotropic agent abrogated the response to CnM but had no effect on a control stimulus that did not require processing. Both aspartic acid and cysteine protease inhibitors blocked effective processing of CnM, so that it was unable to stimulate T cells. Finally, an inhibitor of microfilament polymerization abrogated proliferation to CnM. These results indicate that the mitogenic activity of C. neoformans requires phagocytosis of the organism, lysosomal or endosomal processing, proteolytic activity, and microfilament polymerization and intracellular transport as a prerequisite for T-cell proliferation.

Pseudomonas aeruginosa exoenzyme S induces transcriptional expression of proinflammatory cytokines and chemokines.

Pseudomonas aeruginosa infection of cystic fibrosis patients causes lung damage that is substantially orchestrated by cytokines. In this study, multi-gene probe analysis was used to characterize the ability of the P. aeruginosa mitogen, exoenzyme S, to induce proinflammatory and immunoregulatory cytokines and chemokines. Exoenzyme S strongly induced transcription of proinflammatory cytokines and chemokines (tumor necrosis factor alpha, interleukin-1alpha [IL-1alpha], IL-1beta, IL-6, IL-8, MIP-1alpha, MIP-1beta, MCP-1, RANTES, and I-309), modest transcription of immunoregulatory cytokines (IL-10 and IL-12p40), and weak transcription of Th1 cytokines (IL-2 and gamma interferon). The response occurred early and subsided without evolving over time. These data suggest that cells responding to exoenzyme S would rapidly express proinflammatory cytokines and chemokines that may contribute to pulmonary inflammation in cystic fibrosis.

Accelerated replicative senescence of the peripheral immune system induced by HIV infection.

OBJECTIVES: HIV induces rapid turnover of T lymphocytes but whether this leads to replicative senescence of CD4+ and CD8+ cells and contributes to AIDS symptoms is unclear. The aim of this study was to address this question by analyzing telomere length in blood cell populations as a measure of replicative history in a significant number of patients infected with HIV. DESIGN: Total peripheral blood mononuclear cells (PBMCs), CD4+ or CD8+ cells were isolated from blood collected from a total of 73 HIV patients and 27 controls. Samples were isolated to measure telomere length, telomerase activity and proliferative ability, and analyses were carried out in a blind experimental protocol. METHODS: PBMCs isolated on Ficoll-Hypaque gradients were washed and prepared for additional fractionation into CD4+ and CD8+ cells using antibody-bound magnetic beads. Total PBMCs, CD4+ and CD8+ cells were used for cell cycle analysis, for telomerase activity assays and were measured for telomere length using the terminal restriction fragment assay. RESULTS: Telomere analyses in this study show a clear (P < 0.0001) inverse relationship between telomere length and progression of immunosuppression, with HIV infection resulting in a five-fold or greater acceleration of aging of the circulating PBMC component of the immune system. Patients who are 37 years old showed telomere lengths similar to uninfected 75-year-olds. Telomere loss correlated well with progression of AIDS and with reduced proliferative ability of patient PBMCs but was unrelated to telomerase activity. Mean telomere length was shorter in both CD4+ and CD8+ cells, with three-fold higher rates of telomere loss for CD8+ lymphocytes. CONCLUSIONS: These data provide strong support for the occurrence of accelerated replicative aging of the peripheral immune system, possibly resulting in a loss of T cells leading to AIDS symptoms.

Exoenzyme S from Pseudomonas aeruginosa induces apoptosis in T lymphocytes.

Exoenzyme S from Pseudomonas aeruginosa is a unique T cell mitogen; it is a powerful immunostimulus that activates a large proportion of T cells, but results in delayed and reduced lymphocyte proliferation. This study was performed to explain the discrepancy between early T cell activation and subsequent proliferation. Studies revealed that exoenzyme S induced rapid and unsustained surface expression of CD69, but could not induce interleukin-2 receptor alpha (IL-2R alpha) up-regulation on T cells. IL-2 was undetectable in supernatants and addition of rIL-2 could not reverse the unresponsiveness, indicating that anergy was not involved. Exoenzyme S induced membrane phosphatidylserine translocation, DNA hypodiploidy, and DNA fragmentation, implicating apoptosis as the mechanism for the unresponsiveness. Exoenzyme S-induced apoptosis shows features of both propriocidal and death by neglect, suggesting shared characteristics of an intermediate pathway. Thus, a Pseudomonas exoproduct induces T cell apoptosis, which may contribute to the pathogenesis of Pseudomonas infections in diseases such as cystic fibrosis.

Recombinant Pseudomonas exoenzyme S and exoenzyme S from Pseudomonas aeruginosa DG1 share the ability to stimulate T lymphocyte proliferation.

Exoenzyme S from P. aeruginosa DG1 and recombinant exoenzyme S derived from strain 388 have distinct characteristics, which has led to a controversy about their homology and their pathophysiologic consequences. We have been investigating the ability of exoenzyme S to activate T lymphocytes, and therefore performed studies to determine whether exoenzyme S from P. aeruginosa DG1 and recombinant exoenzyme S derived from strain 388 and expressed in Pseudomonas aeruginosa PA103 or in E. coli BL21(DE3), could induce T lymphocyte activation and proliferation. Both preparations were able to activate T cells and induce lymphocyte proliferation at similar levels as measured by flow cytometry of surface-activation markers and DNA synthesis, respectively. Further, a monoclonal antibody raised against exoenzyme S from strain DG1 partially neutralized T cell activation induced by recombinant exoenzyme S and bound to it in an immunoblot suggesting that the epitope responsible for T cell activation is shared by exoenzyme S from strain DG1 and recombinant exoenzyme S. These data suggest that the two different preparations of exoenzyme S, despite biochemical differences, share the characteristic that is responsible for T lymphocyte activation.

Pseudomonas aeruginosa exoenzyme S stimulates murine lymphocyte proliferation in vitro.

The exuberant immunoinflammatory response that is associated with Pseudomonas aeruginosa infection is the major source of the morbidity and mortality in cystic fibrosis (CF) patients. Previous studies have established that an exoproduct of P. aeruginosa (exoenzyme S) is a mitogen for human T lymphocytes and activates a larger percentage of T cells than most superantigens, which may contribute to the immunoinflammatory response. An animal model would facilitate studies of the pathophysiologic consequences of this activation. As a first step toward developing an animal model, the murine lymphocyte response to exoenzyme S was examined. When stimulated with exoenzyme S, splenocytes isolated from naive mice entered S phase and proliferated. The optimum response occurred after 2 to 3 days in culture, at 4 x 10(5) cells per well and 5.0 micrograms of exoenzyme S per ml. The response was not due to lipopolysaccharide, since Rhodobacter sphaeroides lipid A antagonist did not block the response. Other preparations of exoenzyme S stimulated lymphocyte proliferation, since the response to recombinant exoenzyme S (rHisExo S) cloned from strain 388 was similar to the response to exoenzyme S from strain DG1. There was evidence that genetic variability influenced the response, since A/J, CBA/J, and C57BL/6 mice were high responders and BALB/cJ mice were low responders following stimulation with exoenzyme S. Both splenic T and B lymphocytes entered the cell cycle in response to exoenzyme S. Thus, murine lymphocytes, like human lymphocytes, respond to P. aeruginosa exoenzyme S, which supports the development of a murine model that may facilitate our understanding of the role that exoenzyme S plays in the pathogenesis of P. aeruginosa infections in CF patients.

The capsule of Cryptococcus neoformans reduces T-lymphocyte proliferation by reducing phagocytosis, which can be restored with anticapsular antibody.

Cell-mediated immunity is critical for the host defense to Cryptococcus neoformans, as demonstrated by numerous animal studies and the prevalence of the infection in AIDS patients. Previous studies have established that the polysaccharide capsule contributes to the virulence of C. neoformans by suppressing T-lymphocyte proliferation, which reflects the clonal expansion of T lymphocytes that is a hallmark of cell-mediated immunity. The present studies were performed to identify the major mechanism by which polysaccharide impairs lymphocyte proliferation, since capsular polysaccharide has the potential to affect the development of T-lymphocyte responses by stimulating production of interleukin-10 (IL-10), inhibiting phagocytosis, and inducing shedding of cell surface receptors. We demonstrate that polysaccharide inhibits lymphocyte proliferation predominantly by blocking uptake of C. neoformans, which is crucial for subsequent lymphocyte proliferation. In addition, we show that polysaccharide did not suppress lymphocyte proliferation via an IL-10-dependent mechanism, nor did it affect critical surface receptor interactions on the T cell or antigen-presenting cell. Having established that polysaccharide impairs phagocytosis, we performed studies to determine whether opsonization with human serum or with anticapsular antibody could reverse this effect. Impaired uptake and lymphocyte proliferation that were induced by polysaccharide can be enhanced through opsonization with monoclonal antibodies or human serum, suggesting that antipolysaccharide antibodies might enhance the host defense by restoring uptake of the organism and subsequent presentation to T lymphocytes. These studies support the therapeutic potential of stimulating cell-mediated immunity to C. neoformans with anticapsular antibody.

Interleukin-8 induces lymphocyte chemotaxis into the pleural space. Role of pleural macrophages.

The pleural space is a potential compartment between the lung and chest wall that becomes filled with fluid and inflammatory cells in a number of respiratory diseases. In an attempt to understand one aspect of the inflammatory process in the pleural space, we compared the responses in three different diseases (congestive heart failure [CHF], tuberculosis [TB], and cancer). Large concentrations of interleukin-8 (IL-8) were detected in cancer and TB effusions, but not in CHF. Surprisingly, the concentration of IL-8 correlated best with lymphocyte recruitment and not with neutrophil recruitment. Pleural fluid from cancer and TB patients was chemotactic for lymphocytes, and this activity was partly blocked by an anti-IL-8 antibody in cancer and completely blocked in TB. To determine whether there was the potential for a chemotactic gradient into the pleural space, pleural effusion cells were analyzed for the expression of IL-8. Cells in the effusions of cancer patients expressed IL-8, whereas IL-8 could not be detected from the cells of TB and CHF effusions. To explore the possible role of pleural macrophages in the regulation of IL-8, pleural effusion cells were treated with culture supernatants from stimulated pleural macrophages. Stimulated pleural macrophages were able to induce expression of messenger RNA (mRNA) for IL-8 and IL-8 protein production, and this activity was abrogated by blocking tumor necrosis factor-alpha. These findings suggest that soluble IL-8 is an important factor for the recruitment of lymphocytes into the pleural space, and that this cytokine is produced by both pleural structural and cancer cells after their activation by macrophage-derived, cytokine-mediated signals.

The cell wall and membrane of Cryptococcus neoformans possess a mitogen for human T lymphocytes.

The mechanism of human T-lymphocyte activation by the pathogenic yeast Cryptococcus neoformans has not been established. Previous investigations have suggested that C. neoformans contains a mitogen for T lymphocytes, while other investigators have attributed lymphocyte proliferation in vitro to a recall antigen. Because of the potential importance of the mechanism of T-cell activation for our understanding of the immune response to C. neoformans, the present studies were performed to determine whether C. neoformans contains a mitogen for T lymphocytes. C. neoformans stimulates fetal blood lymphocytes to proliferate and stimulates proliferation of CD45RA+ cells from adults, indicating that it stimulates naive T cells. The T-cell response to C. neoformans was dependent upon the presence of accessory cells. However, allogeneic cells were sufficient for accessory cell function, indicating that the response was not major histocompatibility complex restricted. The percentage of T cells in the cell cycle was higher than that with the recall antigen tetanus toxoid but lower than that with the mitogenic lectin phytohemagglutinin A or the superantigen Staphylococcus enterotoxin B. Precursor frequency analysis established that 1 in 7,750 +/- 2, 270 T cells proliferated in response to the cryptococcal cell wall and membrane. Compared to the case for most mitogens or superantigens, the proliferative response is late and the number of T cells that enter the cell cycle and the precursor frequency are low, indicating that the mitogenic effect is modest. However, the mitogenic effect of C. neoformans should be considered when interpreting the immune response to C. neoformans, since even weak mitogens can have profound effects on host defense.

Host defence to pulmonary mycosis.

OBJECTIVE: To provide a basic understanding of the mechanisms of host defense to pathogenic fungi. This will help physicians understand why some patients are predisposed to fungal infections and update basic scientists on how microbial immunology applies to fungal disease. DATA SOURCES: English articles from 1966 to present were identified from a MEDLINE search. STUDY SELECTION: Articles were identified by a MEDLINE search of 'exp lung/' or 'exp lung diseases/' and 'exp fungi/'. The titles and abstracts were screened to identify articles that contained salient information pertaining to host defense of respiratory mycoses. DATA EXTRACTION: Information was summarized from the articles pertaining to host defense of pulmonary mycosis that had been identified by the MEDLINE search. DATA SYNTHESIS: Fungi represent a unique and highly diverse group of pathogenic organisms that have become an increasingly prevalent cause of life-threatening illness. A worldwide increase in persons with immunodeficiency has been a major contributing factor to the increase in fungal disease. As a result, clinicians are faced with an expanding array of fungal infections that pose diagnostic and therapeutic challenges. The respiratory tract is the route of acquisition for many important fungal infections; thus, understanding the host defense in the lung is an essential component of understanding host defense to fungal disease. With this understanding, fungi may be divided on the basis of the predilection of certain mycosis for specific immune defects. CONCLUSIONS: By separating fungi based on the host immune defects that predispose to disease, in conjunction with traditional divisions based on the geographic distribution of fungi, clinicians are able to focus their diagnostic efforts and to identify fungal pathogens better. In addition, an understanding of the normal host defense mechanisms that serve to control fungal infections is essential to the development of novel antifungal therapies.

Interleukin-15 induces antimicrobial activity after release by Cryptococcus neoformans-stimulated monocytes.

A newly described cytokine, interleukin (IL)-15, shares many activities with IL-2; however, little is known about the stimuli for release of IL-15, and its role in antimicrobial host defense has not previously been demonstrated. This study found that Cryptococcus neoformans is a potent stimulus for the release of biologically active IL-15 from monocytes. Both IL-15 and IL-2 made significant contributions to lymphocyte proliferation and lymphocyte-mediated anticryptococcal activity to encapsulated and acapsular C. neoformans. IL-15 restored lymphocyte proliferation and anticryptococcal activity that had been abrogated by blocking IL-2. IL-15 also enhanced the anticryptococcal activity of lymphocytes but did not enhance the activity of monocytes. This suggests that IL-15 and IL-2 cooperate for lymphocyte activation and proliferation in vitro and demonstrates that IL-15 can induce antimicrobial activity. Taken together, these data suggest that microbes, and in particular C. neoformans, are an important stimulus for IL-15 and that IL-15 may have an important role in induction of antimicrobial effector mechanisms.

Pseudomonas aeruginosa exoenzyme S is a mitogen but not a superantigen for human T lymphocytes.

Virtually all cystic fibrosis (CF) patients become infected with Pseudomonas aeruginosa, and once the infection is established, the organism is rarely cleared. One of the P. aeruginosa virulence factors, exoenzyme S, has been shown to correlate with increased morbidity and mortality both in rat models of chronic pulmonary inflammation and in human CF patients. It has previously been shown that exoenzyme S is a potent stimulus for the proliferation of T cells in greater than 95% of adults, which could contribute to the pathogenesis of CF. The goal of this study was to determine the mechanism of T-cell stimulation by exoenzyme S in an effort to shed light on the immune response and contribute to understanding its role in P. aeruginosa pathogenesis. The current studies demonstrate that exoenzyme S stimulates naive T cells, since fetal blood lymphocytes proliferated and adult lymphocytes that expressed CD45RA proliferated. The percentage of T cells activated by exoenzyme S after a 4-h culture (as measured by CD69 surface expression) was intermediate in magnitude compared to levels induced by a panel of superantigens and mitogens. To determine the mechanism of activation, the requirement for accessory cells was investigated. The proliferative response to exoenzyme S was dependent on the presence of accessory cells but was not blocked by an anti-DR antibody. Exoenzyme S activated both CD4(+) and CD8(+) T cells, but CD4(+) T cells were preferentially activated. The Vbeta repertoire of donor T cells showed no preferential activation or preferential expansion after stimulation by exoenzyme S, suggesting that it is not a superantigen. Taken together, our data suggest that exoenzyme S is a T-cell mitogen but not a superantigen. Activation of a large percentage of T lymphocytes by exoenzyme S may produce a lymphocyte-mediated inflammatory response that should be considered in the pathogenesis of CF.

Both CD4+ and CD8+ human lymphocytes are activated and proliferate in response to Cryptococcus neoformans.

The current studies were performed to determine the contribution of T-cell subsets to lymphocyte proliferation in response to Cryptococcus neoformans, the most common invasive mycosis in acquired immune deficiency syndrome. We demonstrate for the first time that both human CD4 and CD8 cells are activated in response to C. neoformans. Both CD4 and CD8 cells express interleukin-2 receptor alpha (IL-2R alpha) and transferrin receptor and proliferate in response to C, neoformans, however proliferation of CD8 cells was dependent upon CD4 cells. The requirement for CD4 cells was complex, since CD8 enriched cells failed to express mRNA for IL-2, suggesting that CD4-dependent IL-2 production was required for CD8-cell proliferation. However, IL-2 was not sufficient to restore CD8-cell proliferation. These studies provide experimental evidence in humans to support the clinical impression that CD4 cells are important in cryptococcosis, and suggest that the appropriate CD4-derived signals could allow CD8 cells to assist in host defence.

Proteins in the cell wall and membrane of Cryptococcus neoformans stimulate lymphocytes from both adults and fetal cord blood to proliferate.

Cryptococcus neoformans is an encapsulated yeast that infects patients who have defective cell-mediated immunity, including AIDS, but rarely infects individuals who have intact cell-mediated immunity. Studies of the immune response to C. neoformans have been hampered by a paucity of defined T-lymphocyte antigens, and hence, the understanding of the T-cell response is incomplete. The goal of this study was to separate C. neoformans into its component parts, determine whether those components stimulate lymphocyte proliferation, perform preliminary characterization of the proteins, and establish the potential mechanism of lymphocyte proliferation. The lymphocyte response to fungal culture medium, whole organisms, disrupted organisms, and the yeast intracellular fraction or cell wall and membrane was studied by determining thymidine incorporation and by determining the number of lymphocytes at various times after stimulation. The cell wall and membrane of C. neoformans stimulated lymphocyte proliferation, while the intracellular fraction and culture filtrate did not. The optimal response occurred on day 7 of incubation, with 4 x 10(5) peripheral blood mononuclear cells per well and with 13 microg of cryptococcal protein per ml. The number of lymphocytes increased with time in culture, indicating that thymidine incorporation was accompanied by proliferation. Proteinase K treatment of the cell wall and membrane abrogated lymphocyte proliferation, indicating that the molecule was a protein. [35S]methionine labeling, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography were performed to analyze the proteins contained in the cell wall and membrane, intracellular fraction, and culture filtrate. At least 18 discrete bands were resolved from the cell wall and membrane. Since a large percentage of healthy adults responded to the cryptococcal cell wall and membrane, a mitogenic effect was investigated by testing proliferation of fetal cord blood lymphocytes. The percentage of fetal samples that proliferated in response to the cell wall and membrane was similar to the percentage of fetal samples that proliferated in response to Staphylococcus enterotoxin B, a microbial mitogen. Thus, a protein in the cell wall and membrane of C. neoformans is a potent stimulant of lymphocyte proliferation and has mitogenic properties, which may have important implications for cell-mediated immunity to C. neoformans.

Purification and characterization of the double-stranded DNA-activated protein kinase, DNA-PK, from human placenta.

The double-stranded DNA-activated protein kinase (DNA-PK) is a serine-threonine protein kinase that is composed of a large catalytic subunit (p350) and a DNA-binding protein of 70 and 80 kDa subunits known as the Ku autoantigen. When targeted to DNA by free DNA ends, DNA-PK phosphorylates many DNA-binding proteins and transcription factors. Previously, DNA-PK had only been purified and characterized from transformed human tissue culture cells. Here we report that DNA-PK is an abundant protein in human placenta and lymphocytes. We have purified the placental DNA-PK to homogeneity and show that its biochemical properties are similar to those of the HeLa cell enzyme.

Pseudomonas aeruginosa exoenzyme S induces proliferation of human T lymphocytes.

Pseudomonas aeruginosa is a gram-negative bacterium that is responsible for devastating acute and chronic infections, which include bronchiectasis in cystic fibrosis, nosocomial pneumonia, and infection of burn wounds. Previous studies have demonstrated that these patients have impaired host responses, including cell-mediated immune responses, which are important in anti-Pseudomonas host defense. The P. aeruginosa exoproduct, exoenzyme S, has a number of characteristics which suggest that it might be important in cell-mediated immunity. To determine whether exoenzyme S activates lymphocytes to proliferate, peripheral blood mononuclear cells (PBMC) from normal volunteers were stimulated with purified exoenzyme S, and the lymphocyte response was assessed by measuring [3H]thymidine uptake and by counting the number of cells after various times in culture. Ninety-five percent of healthy adult donors had a lymphocyte response to exoenzyme S. The optimal lymphocyte response occurred on day 7, with 4 x 10(5) PBMC per microtiter well when cells were stimulated with 10 micrograms exoenzyme S per ml. [3H]thymidine uptake correlated with an increase in the number of mononuclear cells, indicating that proliferation occurred. In unseparated PBMC, T cells, and to a lesser extent B cells, proliferated. Purified T cells proliferated, while purified B cells proliferated only after the addition of irradiated T cells. Thus, T lymphocytes are necessary and sufficient for the proliferative response to exoenzyme S. We speculate that exoenzyme S from P. aeruginosa is important in T-lymphocyte-mediated host defense to P. aeruginosa. In strategies to enhance impaired cell-mediated immunity, exoenzyme S should be considered as a potential stimulant.

CD8 cells play a critical role in delayed type hypersensitivity to intact Cryptococcus neoformans.

Although cell-mediated immunity is critical for optimal host defense to C. neoformans, the role of T lymphocyte subsets is complex and poorly understood. CD8 cells are important both for optimal host defense against C. neoformans, and for expression of delayed type hypersensitivity (DTH). Because host defense correlates with the ability to mount a DTH response to C. neoformans, the current studies were performed to determine the mechanism by which CD8 cells participate in DTH. Mice were immunized by the intratracheal route with live C. neoformans, or by the subcutaneous route with heat-killed C. neoformans. Mice were depleted of CD8 cells in vivo by administration of mAb. After challenge with soluble cryptococcal Ag, the DTH response was quantified as footpad swelling. We found that mice depleted of CD8 cells before immunization were unable to express DTH. Mice depleted of CD8 cells after immunization but before challenge also were unable to express DTH. Splenocytes of mice depleted of CD8 cells in vivo, before immunization, failed to transfer DTH to naive, undepleted mice. Immune splenocytes depleted of CD8 cells in vitro also failed to transfer DTH to naive, undepleted mice. These data indicate that CD8 cells were necessary during the challenge and immunizing phases of DTH, and were necessary for expression of DTH. However, CD8 cell depletion did not abrogate DTH in mice immunized with either soluble cryptococcal Ag in complete Freund's adjuvant, or sheep red blood cells, which are mediated by CD4 cells. These data suggest that CD8 cells play a critical role in the cell-mediated immune response to C. neoformans. Based on this information, it may be possible to protect hosts with deficiencies of CD4 cells, such as in AIDS, by designing immunizing strategies for stimulating CD8 cells.