me S would rapidly express proinflammatory cytokines and chemokines that may contribute to pulmon... more me S would rapidly express proinflammatory cytokines and chemokines that may contribute to pulmonary inflammation in cystic fibrosis. Virtually all cystic fibrosis (CF) patients are colonized by Pseudomonas aeruginosa, and 90% of those that are colonized die as a result of lung damage (11). One of the hallmarks of the lung damage in CF is an ineffective inflammatory response that results in severe neutrophil-mediated pulmonary damage and an inability to clear the organisms. P. aeruginosa contributes to the lung damage by the production of virulence factors; one of the most important virulence factors, exoenzyme S, has been shown to induce pulmonary damage in animal models (17, 29, 35), and increased levels of exoenzyme S correlate with human disease (18, 36). The cytotoxicity of exoenzyme S for epithelial cells follows both contact-dependent type III translocation into eukaryotic target cells and contact-independent type III secretion, suggesti
Erythroderma is a potentially fatal dermatologic emergency that is often mistaken for infection. ... more Erythroderma is a potentially fatal dermatologic emergency that is often mistaken for infection. Indeed, the fact that it is difficult to diagnosis is the main contributor to its significant mortality rate, as treatment is readily available. We present a case of a 36-year-old man who was incorrectly diagnosed and treated for 2 months. We review the etiologies, initial work-up and management of this disease. In our case, the patient was ill, had lost 11.3 kg and developed systemic inflammatory response syndrome. Without proper treatment he was at risk of developing full-blown sepsis. Although there are many causes of erythroderma, prompt initial treatment directed at the underlying etiology typically results in a rapid remission.
Exoenzyme S from Pseudomonas aeruginosa is a unique T cell mitogen; it is a powerful immunostimul... more 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 ␣ (IL-2R␣) upregulation 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.
The exuberant immunoinflammatory response that is associated with Pseudomonas aeruginosa infectio... more 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 immunoinflam- matory 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 3 105 cells per well and 5.0 mg of exoenzyme S per ml. The response was not due to lipopolysaccharide, since Rhodobacter sphaeroides lipid A antagonist did not block the response. Othe...
The case: A 30-year-old man presented with shortness of breath 5 days after returning from a Hawa... more The case: A 30-year-old man presented with shortness of breath 5 days after returning from a Hawaiian vacation. He reported a 2-week history of progressive difficulty breathing and pain between his shoulder blades, especially when in a supine position. The patient had been previously well, and he was a nonsmoker. The patient described the difficulty breathing as being primarily the inability to take a deep breath. The pain was nonradiating, but it was felt centrally from front to back. He denied having any fever, nighttime sweats, weight loss, cough or exposure to any airborne fumigants. He reported that while on holiday he had been too tired to take part in a number of sporting activities in which he would have normally participated. The patient also reported having had a nonpruritic "rash" on his torso for about 1 week that had spontaneously resolved before he returned to Canada. On examination, the patient was not in distress, his vital signs were normal and he sat on the examination table in a somewhat flexed position. The patient's head and neck examination was unremarkable. Examination of his torso revealed a "band" of telangiectasia on the anterior portion of his lower chest (Figure 1). The patient reported that the band had appeared overnight, and that it was the same as the rash that had resolved 2 weeks earlier. The results of both cardiac (including electrocardiography) and respiratory examinations were unremarkable. In the supine position, the patient's respirations were exaggerated, and he noticeably used his chest and abdomen for inspiration. A radiograph showed a large anterior mediastinal mass and normal lungs and pleura (Figure 2). The results of a CT scan showed a heterogenous anterior mediastinal mass that measured 13 × 9 × 11 cm (lateral × anteroposterior × cranio-caudal) (Figure 3). The mass
Cell-mediated immunity is critical for the host defense to Cryptococcus neoformans, as demonstrat... more 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.
Virtually all cystic fibrosis (CF) patients become infected with Pseudomonas aeruginosa, and once... more 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 V 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. Pseudomonas aeruginosa is an opportunistic organism of immunocompromised individuals (8). P. aeruginosa can cause both acute and chronic infections such as those seen in nosocomial pneumonia, burn wounds, septicemia, and bronchiectasis in cystic fibrosis (CF) patients. Bronchopulmonary infections are the primary cause of morbidity and mortality in CF patients (34). It has been shown that the vast majority of CF patients by the age of 11 become colonized with P. aeruginosa (25) and that once established, this infection is rarely cleared (37). One of the P. aeruginosa virulence factors associated with disease is exoenzyme S (15, 16, 47). In this study, we have investigated the effect of P. aeruginosa exoenzyme S on T-cell activation and proliferation. Exoenzyme S is a 49-to 53-kDa protein that is capable of transferring an ADP-ribose to various proteins in vitro (9) and stimulates human T cells to proliferate (32). Approximately 90% of clinical isolates from CF patients produce exoenzyme S, and 40% of the isolates produce the enzymatically active form (48). Moreover, increases in morbidity and mortality rates have been associated with exoenzyme S-producing strains compared to isogenic mutants in a rat model of chronic P. aeruginosa infection (47). Screening of clinical isolates among CF patients chronically colonized with P. aeruginosa showed that acute deterioration of pulmonary function was accompanied by increased exoenzyme S production (16). Despite the inability of antibiotics to completely eliminate P. aeruginosa from the airways of CF patients during pulmonary exacerba
Exoenzyme S from P. aeruginosa DG1 and recombinant exoenzyme S derived from strain 388 have disti... more 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...
Pseudomonas aeruginosa infection of cystic fibrosis patients causes lung damage that is substanti... more 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-1␣ [IL-1␣], IL-1, IL-6, IL-8, MIP-1␣, MIP-1, 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.
me S would rapidly express proinflammatory cytokines and chemokines that may contribute to pulmon... more me S would rapidly express proinflammatory cytokines and chemokines that may contribute to pulmonary inflammation in cystic fibrosis. Virtually all cystic fibrosis (CF) patients are colonized by Pseudomonas aeruginosa, and 90% of those that are colonized die as a result of lung damage (11). One of the hallmarks of the lung damage in CF is an ineffective inflammatory response that results in severe neutrophil-mediated pulmonary damage and an inability to clear the organisms. P. aeruginosa contributes to the lung damage by the production of virulence factors; one of the most important virulence factors, exoenzyme S, has been shown to induce pulmonary damage in animal models (17, 29, 35), and increased levels of exoenzyme S correlate with human disease (18, 36). The cytotoxicity of exoenzyme S for epithelial cells follows both contact-dependent type III translocation into eukaryotic target cells and contact-independent type III secretion, suggesti
Erythroderma is a potentially fatal dermatologic emergency that is often mistaken for infection. ... more Erythroderma is a potentially fatal dermatologic emergency that is often mistaken for infection. Indeed, the fact that it is difficult to diagnosis is the main contributor to its significant mortality rate, as treatment is readily available. We present a case of a 36-year-old man who was incorrectly diagnosed and treated for 2 months. We review the etiologies, initial work-up and management of this disease. In our case, the patient was ill, had lost 11.3 kg and developed systemic inflammatory response syndrome. Without proper treatment he was at risk of developing full-blown sepsis. Although there are many causes of erythroderma, prompt initial treatment directed at the underlying etiology typically results in a rapid remission.
Exoenzyme S from Pseudomonas aeruginosa is a unique T cell mitogen; it is a powerful immunostimul... more 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 ␣ (IL-2R␣) upregulation 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.
The exuberant immunoinflammatory response that is associated with Pseudomonas aeruginosa infectio... more 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 immunoinflam- matory 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 3 105 cells per well and 5.0 mg of exoenzyme S per ml. The response was not due to lipopolysaccharide, since Rhodobacter sphaeroides lipid A antagonist did not block the response. Othe...
The case: A 30-year-old man presented with shortness of breath 5 days after returning from a Hawa... more The case: A 30-year-old man presented with shortness of breath 5 days after returning from a Hawaiian vacation. He reported a 2-week history of progressive difficulty breathing and pain between his shoulder blades, especially when in a supine position. The patient had been previously well, and he was a nonsmoker. The patient described the difficulty breathing as being primarily the inability to take a deep breath. The pain was nonradiating, but it was felt centrally from front to back. He denied having any fever, nighttime sweats, weight loss, cough or exposure to any airborne fumigants. He reported that while on holiday he had been too tired to take part in a number of sporting activities in which he would have normally participated. The patient also reported having had a nonpruritic "rash" on his torso for about 1 week that had spontaneously resolved before he returned to Canada. On examination, the patient was not in distress, his vital signs were normal and he sat on the examination table in a somewhat flexed position. The patient's head and neck examination was unremarkable. Examination of his torso revealed a "band" of telangiectasia on the anterior portion of his lower chest (Figure 1). The patient reported that the band had appeared overnight, and that it was the same as the rash that had resolved 2 weeks earlier. The results of both cardiac (including electrocardiography) and respiratory examinations were unremarkable. In the supine position, the patient's respirations were exaggerated, and he noticeably used his chest and abdomen for inspiration. A radiograph showed a large anterior mediastinal mass and normal lungs and pleura (Figure 2). The results of a CT scan showed a heterogenous anterior mediastinal mass that measured 13 × 9 × 11 cm (lateral × anteroposterior × cranio-caudal) (Figure 3). The mass
Cell-mediated immunity is critical for the host defense to Cryptococcus neoformans, as demonstrat... more 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.
Virtually all cystic fibrosis (CF) patients become infected with Pseudomonas aeruginosa, and once... more 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 V 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. Pseudomonas aeruginosa is an opportunistic organism of immunocompromised individuals (8). P. aeruginosa can cause both acute and chronic infections such as those seen in nosocomial pneumonia, burn wounds, septicemia, and bronchiectasis in cystic fibrosis (CF) patients. Bronchopulmonary infections are the primary cause of morbidity and mortality in CF patients (34). It has been shown that the vast majority of CF patients by the age of 11 become colonized with P. aeruginosa (25) and that once established, this infection is rarely cleared (37). One of the P. aeruginosa virulence factors associated with disease is exoenzyme S (15, 16, 47). In this study, we have investigated the effect of P. aeruginosa exoenzyme S on T-cell activation and proliferation. Exoenzyme S is a 49-to 53-kDa protein that is capable of transferring an ADP-ribose to various proteins in vitro (9) and stimulates human T cells to proliferate (32). Approximately 90% of clinical isolates from CF patients produce exoenzyme S, and 40% of the isolates produce the enzymatically active form (48). Moreover, increases in morbidity and mortality rates have been associated with exoenzyme S-producing strains compared to isogenic mutants in a rat model of chronic P. aeruginosa infection (47). Screening of clinical isolates among CF patients chronically colonized with P. aeruginosa showed that acute deterioration of pulmonary function was accompanied by increased exoenzyme S production (16). Despite the inability of antibiotics to completely eliminate P. aeruginosa from the airways of CF patients during pulmonary exacerba
Exoenzyme S from P. aeruginosa DG1 and recombinant exoenzyme S derived from strain 388 have disti... more 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...
Pseudomonas aeruginosa infection of cystic fibrosis patients causes lung damage that is substanti... more 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-1␣ [IL-1␣], IL-1, IL-6, IL-8, MIP-1␣, MIP-1, 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.
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