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Evaluation of NKT Cells in Leishmaniasis
Abstracts S95
J ALLERGY CLIN IMMUNOL VOLUME 119, NUMBER 1
Evaluation of NKT Cells in Leishmaniasis R. A. Campos, J. M. Cunha, O. F. Jesus, O. Bacellar, F. C. C. Figueiredo, R. G. D. Lima, R. S. Matos, E. M. Carvalho; Universidade Federal da Bahia, Salvador-BA, BRAZIL. RATIONALE: Th1 response is protective in Leishmaniasis. The mucosa subtype is more agressive than the cutaneous subtype affecting the upper respiratory tract with destructive lesions. NKT cells are involved in the innate immune response and produce both Th1 and Th2 cytokines. CD41 NKT cells release mainly Th2 cytokines. There are experimental evidences of the participation of these cells in Leismaniasis but no data in humans. METHODS: We studied controls, patients with cutaneous and mucosal Leishmaniasis in an endemic area in Brazil. Using flow cytometry, we analized the NKT cells and the intracellular production of cytokines by these cells from the peripheral blood mononuclear cells with fluorocrome conjugated monoclonal antibodies: anti-6B11, anti-CD3, anti-CD4, anti-Va24, anti-interferon-g e anti-interleucina 210. NK T cells were defined by the double expression of CD3 and 6B11 or CD3 and Va24. RESULTS: 10 controls, 9 pacients with cutaneous subtype and 4 with mucosal subtype were evaluated. The NK T cells were lower in the mucosal subtype than the controls (1,23%60,5 vs. 0,16%60,05; p50,01) and the cutaneous subtype (0,73%60,18 vs. 0,16%60,05; p50,02). There was no significative difference in the intracellular production of cytokines although with higher percentages in the mucosal subtype. CD4 NK T cells were found mostly in the mucosal subtype but not reaching statistical significance (47,5%69.8 vs 36,1% 6 6.8) CONCLUSIONS: The NK T cells may be associated with the protective Th1 response in Leishmaniasis through the production of interferon-g since the mucosal subtype, more agressive, showed fewer proportions of these cells. Funding: FAPESB
373
B Cell Superantigen-induced Immune Complex Lung Inflammation A. L. Anderson, A. I. Levinson; University of Pennsylvania, Philadelphia, PA. RATIONALE: Peptostreptococcus magnus Protein L functions as a B cell superantigen (SAg) by binding to Vkappa light chain determinants on many human and mouse Igs, irrespective of their antigen specificities. The ability of B cell SAgs to bind to a large amount of serum IgG underscores their potential to cause immune complex-mediated tissue injury. We investigated whether such a B cell SAg elicits inflammation when introduced into the airways and, if so, whether the reaction resembles that elicited by immune complexes. METHODS: C57BL/6 mice were administered either Protein L or BSA intratracheally (i.t.). Bronchoalveolar lavage fluid (BALF) was collected at various time points and analyzed for total neutrophil counts. Simultaneously obtained lung samples were analyzed for myeloperoxidase (MPO) and histology. Studies were repeated in mast cell deficient mice and wildtype littermates to investigate potential contributions of mast cells. RESULTS: Protein L-treated mice accumulated neutrophils in their BALF peaking at 24 hours after i.t. challenge. Their lungs showed margination of leukocytes in blood vessel lumens and a perivascular infiltrate of inflammatory cells. These changes were accompanied by diapedesis of erythrocytes and, in some areas, frank hemorrhage. They were associated with markedly increased tissue concentrations of MPO and were preserved in mast cell deficient mice. CONCLUSIONS: A B cell SAg can induce mast cell-independent inflammation in the lungs with features suggestive of immune complex mediated injury. Since most B cell SAgs are derived from ubiquitous microbes, these results may help elucidate how infectious organisms contribute to the pathogenesis of immune complex mediated lung injury. Funding: NIAID
374
Long-term Clinical Outcomes Following Vaccinia Associated Myopericarditis M. R. Nelson1, L. C. Collins1, J. E. Atwood1, R. E. Eckert2, E. A. Shry2, D. J. Shelton1, W. Scott1, R. J. M. Engler1; 1Walter Reed Army Medical Center, Washington, DC, 2Brooke Army Medical Center, Fort Sam Houston, TX. RATIONALE: Approximately 115 cases of MP have been identified during the ongoing DoD smallpox vaccination program. There is little known regarding the long-term clinical outcome of Vaccinia associated myopericarditis (VAMP) and the risk of late onset sequelae arising from the acute inflammation. METHODS: A registry of all DoD VAMP patients maintained by the Walter Reed National Vaccine Healthcare Center Network was analyzed for clinical outcomes. Study entry criteria included adequate follow-up data at 6-18 and/or 18-24 months. Outcome measures included any vaccine related symptoms, ECG, graded exercise test (GXT), cardiac imaging (Echo, MRI) and cardiac enzymes. RESULTS: There were 2 deaths during acute VAMP presentation. 53 of the remaining 113 patients met entry criteria. 45/53 (85%) had no VAMP related symptoms beyond 6 months. 8/53 (15%) reported symptoms of mild intermittent chest pain/tightness, palpitations and/or dyspnea. Objective findings did not correlate with symptoms and included 5 (9.4%) minor ECG abnormalities, 2 transient densities on cardiac imaging (1 Echo, 1 MRI), 1 abnormal GXT with normal perfusion study and no abnormal cardiac enzyme tests. The ejection fraction was normal in all examined. CONCLUSIONS: The vast majority of VAMP patients are asymptomatic by 6 months and have preserved systolic function. In addition to the risk of rare early death, some VAMP patients have symptoms that persist beyond 6-18 months that affect quality of life. Minor ECG and cardiac imaging abnormalities may also be present beyond 6 months but rarely correlate with symptoms. Further study is needed to assess longer term outcomes. Funding: Walter Reed Army Medical Center & Vaccine Healthcare Center
375
Does Early BCG (Tokyo Strain) Vaccination have Potency to Prevent Allergy in Infants? R. Ito, H. Saito, K. Matsumoto; National Research Institute for Child Health & Development, Tokyo, JAPAN. RATIONALE: Bacille Calmette Gue´rin (BCG) acts as a strong Th1 adjuvant both in adults in vitro and in murine models and thus, BCG vaccine is supposed to be preventive for allergic diseases. However, previous epidemiological studies in humans have shown no or a marginal effect of BCG on allergic sensitization, presumably because of the differences of races, vaccine strains or timing of vaccination. BCG Tokyo strain inoculated all Japanese infants is known to exert strong immunogenic activities as it induces granulomatous lesions when administered sub-cutaneously. In the present study, we investigated the effect of BCG Tokyo strain on cytokine production profiles in cord blood mononuclear cells (CBMC) in vitro. METHODS: CBMC were isolated from normal full-term deliveries and cultured with two distinct types of BCG (Tokyo and Danish) or PGN for 6 hours. The mRNA expression of TNFa, IFNg, IL-15, IL-12p40, IP-10, IL-10 and IFIT-1 was measured by a quantitative real-time PCR assay. RESULTS: Both BCG strains significantly enhanced the mRNA expression of IFNg, IP-10 and IL-12p40, although the magnitude was far smaller than that by PGN. There were no remarkable differences in the cytokine expression profiles between 2 BCG strains. They failed to induce the mRNA expression of IL-15, IL-10, TNFa and IFIT-1. CONCLUSIONS: (1) The impaired induction of IFIT-1, a sensitive type I IFN-inducible gene, suggests that these BCG strains stimulate CBMC only through TLR2 but not other TLRs. (2) Our results suggest that BCG vaccination may not have enough potency to induce strong Th1-skewing or prevent allergic sensitization in infants. Funding: National Institute of Biomedical Innovation, Japanese Ministry of Education, Culture, Sports, Science and Technology
SUNDAY
372
J ALLERGY CLIN IMMUNOL VOLUME 119, NUMBER 1
Evaluation of NKT Cells in Leishmaniasis R. A. Campos, J. M. Cunha, O. F. Jesus, O. Bacellar, F. C. C. Figueiredo, R. G. D. Lima, R. S. Matos, E. M. Carvalho; Universidade Federal da Bahia, Salvador-BA, BRAZIL. RATIONALE: Th1 response is protective in Leishmaniasis. The mucosa subtype is more agressive than the cutaneous subtype affecting the upper respiratory tract with destructive lesions. NKT cells are involved in the innate immune response and produce both Th1 and Th2 cytokines. CD41 NKT cells release mainly Th2 cytokines. There are experimental evidences of the participation of these cells in Leismaniasis but no data in humans. METHODS: We studied controls, patients with cutaneous and mucosal Leishmaniasis in an endemic area in Brazil. Using flow cytometry, we analized the NKT cells and the intracellular production of cytokines by these cells from the peripheral blood mononuclear cells with fluorocrome conjugated monoclonal antibodies: anti-6B11, anti-CD3, anti-CD4, anti-Va24, anti-interferon-g e anti-interleucina 210. NK T cells were defined by the double expression of CD3 and 6B11 or CD3 and Va24. RESULTS: 10 controls, 9 pacients with cutaneous subtype and 4 with mucosal subtype were evaluated. The NK T cells were lower in the mucosal subtype than the controls (1,23%60,5 vs. 0,16%60,05; p50,01) and the cutaneous subtype (0,73%60,18 vs. 0,16%60,05; p50,02). There was no significative difference in the intracellular production of cytokines although with higher percentages in the mucosal subtype. CD4 NK T cells were found mostly in the mucosal subtype but not reaching statistical significance (47,5%69.8 vs 36,1% 6 6.8) CONCLUSIONS: The NK T cells may be associated with the protective Th1 response in Leishmaniasis through the production of interferon-g since the mucosal subtype, more agressive, showed fewer proportions of these cells. Funding: FAPESB
373
B Cell Superantigen-induced Immune Complex Lung Inflammation A. L. Anderson, A. I. Levinson; University of Pennsylvania, Philadelphia, PA. RATIONALE: Peptostreptococcus magnus Protein L functions as a B cell superantigen (SAg) by binding to Vkappa light chain determinants on many human and mouse Igs, irrespective of their antigen specificities. The ability of B cell SAgs to bind to a large amount of serum IgG underscores their potential to cause immune complex-mediated tissue injury. We investigated whether such a B cell SAg elicits inflammation when introduced into the airways and, if so, whether the reaction resembles that elicited by immune complexes. METHODS: C57BL/6 mice were administered either Protein L or BSA intratracheally (i.t.). Bronchoalveolar lavage fluid (BALF) was collected at various time points and analyzed for total neutrophil counts. Simultaneously obtained lung samples were analyzed for myeloperoxidase (MPO) and histology. Studies were repeated in mast cell deficient mice and wildtype littermates to investigate potential contributions of mast cells. RESULTS: Protein L-treated mice accumulated neutrophils in their BALF peaking at 24 hours after i.t. challenge. Their lungs showed margination of leukocytes in blood vessel lumens and a perivascular infiltrate of inflammatory cells. These changes were accompanied by diapedesis of erythrocytes and, in some areas, frank hemorrhage. They were associated with markedly increased tissue concentrations of MPO and were preserved in mast cell deficient mice. CONCLUSIONS: A B cell SAg can induce mast cell-independent inflammation in the lungs with features suggestive of immune complex mediated injury. Since most B cell SAgs are derived from ubiquitous microbes, these results may help elucidate how infectious organisms contribute to the pathogenesis of immune complex mediated lung injury. Funding: NIAID
374
Long-term Clinical Outcomes Following Vaccinia Associated Myopericarditis M. R. Nelson1, L. C. Collins1, J. E. Atwood1, R. E. Eckert2, E. A. Shry2, D. J. Shelton1, W. Scott1, R. J. M. Engler1; 1Walter Reed Army Medical Center, Washington, DC, 2Brooke Army Medical Center, Fort Sam Houston, TX. RATIONALE: Approximately 115 cases of MP have been identified during the ongoing DoD smallpox vaccination program. There is little known regarding the long-term clinical outcome of Vaccinia associated myopericarditis (VAMP) and the risk of late onset sequelae arising from the acute inflammation. METHODS: A registry of all DoD VAMP patients maintained by the Walter Reed National Vaccine Healthcare Center Network was analyzed for clinical outcomes. Study entry criteria included adequate follow-up data at 6-18 and/or 18-24 months. Outcome measures included any vaccine related symptoms, ECG, graded exercise test (GXT), cardiac imaging (Echo, MRI) and cardiac enzymes. RESULTS: There were 2 deaths during acute VAMP presentation. 53 of the remaining 113 patients met entry criteria. 45/53 (85%) had no VAMP related symptoms beyond 6 months. 8/53 (15%) reported symptoms of mild intermittent chest pain/tightness, palpitations and/or dyspnea. Objective findings did not correlate with symptoms and included 5 (9.4%) minor ECG abnormalities, 2 transient densities on cardiac imaging (1 Echo, 1 MRI), 1 abnormal GXT with normal perfusion study and no abnormal cardiac enzyme tests. The ejection fraction was normal in all examined. CONCLUSIONS: The vast majority of VAMP patients are asymptomatic by 6 months and have preserved systolic function. In addition to the risk of rare early death, some VAMP patients have symptoms that persist beyond 6-18 months that affect quality of life. Minor ECG and cardiac imaging abnormalities may also be present beyond 6 months but rarely correlate with symptoms. Further study is needed to assess longer term outcomes. Funding: Walter Reed Army Medical Center & Vaccine Healthcare Center
375
Does Early BCG (Tokyo Strain) Vaccination have Potency to Prevent Allergy in Infants? R. Ito, H. Saito, K. Matsumoto; National Research Institute for Child Health & Development, Tokyo, JAPAN. RATIONALE: Bacille Calmette Gue´rin (BCG) acts as a strong Th1 adjuvant both in adults in vitro and in murine models and thus, BCG vaccine is supposed to be preventive for allergic diseases. However, previous epidemiological studies in humans have shown no or a marginal effect of BCG on allergic sensitization, presumably because of the differences of races, vaccine strains or timing of vaccination. BCG Tokyo strain inoculated all Japanese infants is known to exert strong immunogenic activities as it induces granulomatous lesions when administered sub-cutaneously. In the present study, we investigated the effect of BCG Tokyo strain on cytokine production profiles in cord blood mononuclear cells (CBMC) in vitro. METHODS: CBMC were isolated from normal full-term deliveries and cultured with two distinct types of BCG (Tokyo and Danish) or PGN for 6 hours. The mRNA expression of TNFa, IFNg, IL-15, IL-12p40, IP-10, IL-10 and IFIT-1 was measured by a quantitative real-time PCR assay. RESULTS: Both BCG strains significantly enhanced the mRNA expression of IFNg, IP-10 and IL-12p40, although the magnitude was far smaller than that by PGN. There were no remarkable differences in the cytokine expression profiles between 2 BCG strains. They failed to induce the mRNA expression of IL-15, IL-10, TNFa and IFIT-1. CONCLUSIONS: (1) The impaired induction of IFIT-1, a sensitive type I IFN-inducible gene, suggests that these BCG strains stimulate CBMC only through TLR2 but not other TLRs. (2) Our results suggest that BCG vaccination may not have enough potency to induce strong Th1-skewing or prevent allergic sensitization in infants. Funding: National Institute of Biomedical Innovation, Japanese Ministry of Education, Culture, Sports, Science and Technology
SUNDAY
372