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The Adaptive Immune Response in Cutaneous Methicillin-Resistant Staphylococcus Aureus Infections
AB230 Abstracts
887
Clinical Features Of The Relationship Between IgG4 And Sclerosing Lesions M. Tatewaki, K. Kurasawa, H. Hirata, M. Watanabe, R. Maezawa, K. Sugiyama, Y. Fukushima, T. Fukuda; Dokkyo University School of Medicine, Mibuchi, JAPAN. RATIONALE: IgG4-related diseases are associated with autoimmune pancreatitis, sclerosing sialadenitis, retroperitoneal fibrosis, and sclerosing cholangitis. Such diseases are characterized by high serum IgG4 concentrations, sclerosing inflammation with numerous IgG4-positive plasma cells. METHODS: The relationship between high IgG4 concentrations with or without sclerosing lesions (Groups A and B, respectively), and normal range of IgG4 concentrations with sclerosing lesions (Group C). Of the 108 patients who had their serum IgG4 concentration measured, we retrospectively classified 24 (18 men, 6 women) in Group A, 21 (10 men, 11 women) in Group B, and 8 (4 men, 4 women) in Group C as having IgG4-related diseases. RESULTS: The mean age of was 65.9, 63.9, and 60.0 years for Groups A, B, and C, respectively. The mean concentration of IgG4 and ratio of IgG4/ IgG in Groups A and B were 6336649 mg/dl vs 3386330 mg/dl and 22.8610.8% vs 10.462.0%, respectively. Furthermore, there was a relationship between IgG4 concentration and number of sclerosing lesions. Complications in Group B were autoimmune diseases (n510), allergic diseases (n56), and Castlemann’s disease (n52). Moreover, as to the cause of the diseases, multiple lymphadenopathy and interstitial pneumonia were detected in Group A (55% and 29%, respectively) and Group B (33% and 48%, respectively). On the other hand, 3 of the 8 patients in Group C who had retroperitoneal fibrosis subsequently developed cancer. CONCLUSIONS: Patients with high IgG4 concentrations had inflammation of multiple organs and immune disorders regardless of the presence of sclerosing lesions. Additionally, retroperitoneal fibrosis with normal IgG4 concentrations may have some involvement in paraneoplastic syndrome.
888
TUESDAY
G Protein Coupled Receptor Kinase 3 (GRK3) Negatively Regulates CXCL12/CXCR4 Signaling and Tumor Migration in Breast Cancer D. J. Fitzhugh, M. W. McGinnis, R. Timoshchenko, R. Lininger, N. Demore, J. Serody, T. K. Tarrant; University of North Carolina, Chapel Hill, NC. RATIONALE: CXCL12/CXCR4 signaling is important in tumor metastasis, but intracellular regulators of CXCR4 in cancer are poorly understood. G protein coupled receptor kinases (GRKs) desensitize and internalize chemokine receptors, and GRK subtype 3 serves as a negative regulator of CXCR4 signaling in WHIM syndrome. Consequently, we investigated whether modulation of GRK3 affected breast cancer metastasis. METHODS: GRK3 and CXCR4 were quantified by real-time PCR in a panel of human breast cancer cell lines. GRK3 levels were manipulated (shRNA knockdown or transient overexpression), and chemotaxis and chemoinvasion were quantified in the modified lines. CXCR4 internalization was determined by flow cytometry after CXCL12 stimulation in vitro. Control or GRK3-specific shRNA was transfected into a luciferase-tagged murine breast cancer line (66c14-luc), which was surgically implanted into susceptible BALB-c mice and serially imaged over time. RESULTS: The CXCR4:GRK3 expression ratio corresponded to metastatic potential in human breast cancer lines (12.1 in MDA-MB-231 vs. 0.005 in MDA-MB-468, p<0.0001). GRK3 knockdown in MDA-MB468 (a nonmetastatic line) had 2.8 fold greater chemotaxis (p50.03) and 3.4 fold greater chemoinvasion (p50.03) to CXCL12 compared to controls. Conversely, GRK3 overexpression in MDA-MB-231 (a highly metastatic line) decreased CXCL12-mediated chemotaxis 2.9 fold (p50.005) and chemoinvasion 5.0 fold (p50.007). GRK3 deficiency impaired CXCR4 internalization (p50.03) in breast cancer cells. In mice, GRK3-deficient 66c14-luc cells showed metastasis to distant sites whereas control cells showed predominantly primary tumor growth.
J ALLERGY CLIN IMMUNOL FEBRUARY 2011
CONCLUSIONS: GRK3 serves as a negative regulator of CXCL12/ CXCR4 in breast cancer, and the CXCR4:GRK3 expression ratio may have prognostic significance for the metastatic potential of breast cancer.
889
The Adaptive Immune Response in Cutaneous MethicillinResistant Staphylococcus Aureus Infections I. A. Myles, C. Fowler, P. Valdez, S. Datta; The National Institutes of Health, Bethesda, MD. RATIONALE: Cutaneous methicillin-resistant staphylococcus aureus (MRSA) infection is an increasingly prevalent clinical dilemma. While it is known that innate immunity and antimicrobial peptides are central in the response to MRSA skin infections, far less is known about the role of adaptive immunity. METHODS: To assess the effect of adaptive immunity we compared lesion size groups of C57BL/6 mice with: 1) two intradermal MRSA inoculations, 2) injection with diluent followed by MRSA inoculation, and 3) use of a CD4 depleting antibody prior to sequential MRSA inoculations, with each group’s inoculations spaced 21-28 days apart. Blood was periodically assessed for anti-MRSA antibodies. On day 27-34 splenocytes and draining lymph nodes were harvested and staphylococcus-specific cytokines and VBeta usage were evaluated. RESULTS: We found that neither prior MRSA exposure nor CD4 depletion had any impact on lesion area. There was no shift in VBeta usage with either singular or sequential infections. There was a significant increase in total anti-MRSA IgG, IgG1, IgG2c, and IgAwith repeat infection. CD4 depletion greatly reduced total IgG and IgG2c but not IgG1 or IgA. We found no measurable IgE. For cytokines, only IL-17 in the draining lymph nodes was significantly higher with repeat exposure. Several other cytokines were independent of exposure history, whereas splenic IL-13 was higher with initial infection. CONCLUSIONS: These results indicate that while an adaptive response is seen with MRSA infections, antibody titers, CD4 status, and T cell cytokine production do not correlate with infection outcome. (Further work on mRNA expression and histology is ongoing.)
890
Response to Cutaneous Immunization with Low Molecular Weight Subunit Keyhole Limpet Hemocyanin (KLH) H. Milgrom1, K. L. Kesler2, M. Byron2, R. J. Harbeck1, R. Holliday2, D. Y. Leung1; 1National Jewish Health, Denver, CO, 2Rho Inc, Chapel Hill, NC. RATIONALE: This study was carried out to determine whether humoral and cellular immunological responses would be provoked by cutaneous immunization with KLH. METHODS: This was an unblinded, single-center, 8-week pilot study in healthy, nonatopic young adults. Twenty-five subjects were randomized; 24 completed the study. Subjects were assigned to 4 groups. Subjects were immunized twice, 3 weeks apart by 1 of 2 routes, scarification (SS) or intradermally (ID), with 1 of 2 doses, 50 mg (3 jabs/1 loaded bifurcated needle) or 250 mg (15 jabs/ 5 needles) for SS and 100 mg or 250 mg for the ID route. Immunoglobulins were measured at baseline and 3 weeks after each immunization. DTH testing was performed at baseline and 3 weeks after the second immunization. RESULTS: Immune responses were detected in all 4 study groups (SS low, SS high, ID low, ID high). Anti-KLH IgG, IgA, and IgM production and the number of subjects responding were greater for high dose ID than for low dose ID. Results for anti-KLH IgG1-3 showed responses to both ID and SS. One subject in each treatment group showed a positive DTH result. The percentages of subjects who doubled their antibody were similar across the 4 study groups. CONCLUSIONS: We show here that it is possible to induce both humoral and cellular immune responses in healthy subjects by either ID or SS administration of KLH. This approach may be useful in studying mechanisms of immune responses in allergic skin diseases such as atopic dermatitis.
887
Clinical Features Of The Relationship Between IgG4 And Sclerosing Lesions M. Tatewaki, K. Kurasawa, H. Hirata, M. Watanabe, R. Maezawa, K. Sugiyama, Y. Fukushima, T. Fukuda; Dokkyo University School of Medicine, Mibuchi, JAPAN. RATIONALE: IgG4-related diseases are associated with autoimmune pancreatitis, sclerosing sialadenitis, retroperitoneal fibrosis, and sclerosing cholangitis. Such diseases are characterized by high serum IgG4 concentrations, sclerosing inflammation with numerous IgG4-positive plasma cells. METHODS: The relationship between high IgG4 concentrations with or without sclerosing lesions (Groups A and B, respectively), and normal range of IgG4 concentrations with sclerosing lesions (Group C). Of the 108 patients who had their serum IgG4 concentration measured, we retrospectively classified 24 (18 men, 6 women) in Group A, 21 (10 men, 11 women) in Group B, and 8 (4 men, 4 women) in Group C as having IgG4-related diseases. RESULTS: The mean age of was 65.9, 63.9, and 60.0 years for Groups A, B, and C, respectively. The mean concentration of IgG4 and ratio of IgG4/ IgG in Groups A and B were 6336649 mg/dl vs 3386330 mg/dl and 22.8610.8% vs 10.462.0%, respectively. Furthermore, there was a relationship between IgG4 concentration and number of sclerosing lesions. Complications in Group B were autoimmune diseases (n510), allergic diseases (n56), and Castlemann’s disease (n52). Moreover, as to the cause of the diseases, multiple lymphadenopathy and interstitial pneumonia were detected in Group A (55% and 29%, respectively) and Group B (33% and 48%, respectively). On the other hand, 3 of the 8 patients in Group C who had retroperitoneal fibrosis subsequently developed cancer. CONCLUSIONS: Patients with high IgG4 concentrations had inflammation of multiple organs and immune disorders regardless of the presence of sclerosing lesions. Additionally, retroperitoneal fibrosis with normal IgG4 concentrations may have some involvement in paraneoplastic syndrome.
888
TUESDAY
G Protein Coupled Receptor Kinase 3 (GRK3) Negatively Regulates CXCL12/CXCR4 Signaling and Tumor Migration in Breast Cancer D. J. Fitzhugh, M. W. McGinnis, R. Timoshchenko, R. Lininger, N. Demore, J. Serody, T. K. Tarrant; University of North Carolina, Chapel Hill, NC. RATIONALE: CXCL12/CXCR4 signaling is important in tumor metastasis, but intracellular regulators of CXCR4 in cancer are poorly understood. G protein coupled receptor kinases (GRKs) desensitize and internalize chemokine receptors, and GRK subtype 3 serves as a negative regulator of CXCR4 signaling in WHIM syndrome. Consequently, we investigated whether modulation of GRK3 affected breast cancer metastasis. METHODS: GRK3 and CXCR4 were quantified by real-time PCR in a panel of human breast cancer cell lines. GRK3 levels were manipulated (shRNA knockdown or transient overexpression), and chemotaxis and chemoinvasion were quantified in the modified lines. CXCR4 internalization was determined by flow cytometry after CXCL12 stimulation in vitro. Control or GRK3-specific shRNA was transfected into a luciferase-tagged murine breast cancer line (66c14-luc), which was surgically implanted into susceptible BALB-c mice and serially imaged over time. RESULTS: The CXCR4:GRK3 expression ratio corresponded to metastatic potential in human breast cancer lines (12.1 in MDA-MB-231 vs. 0.005 in MDA-MB-468, p<0.0001). GRK3 knockdown in MDA-MB468 (a nonmetastatic line) had 2.8 fold greater chemotaxis (p50.03) and 3.4 fold greater chemoinvasion (p50.03) to CXCL12 compared to controls. Conversely, GRK3 overexpression in MDA-MB-231 (a highly metastatic line) decreased CXCL12-mediated chemotaxis 2.9 fold (p50.005) and chemoinvasion 5.0 fold (p50.007). GRK3 deficiency impaired CXCR4 internalization (p50.03) in breast cancer cells. In mice, GRK3-deficient 66c14-luc cells showed metastasis to distant sites whereas control cells showed predominantly primary tumor growth.
J ALLERGY CLIN IMMUNOL FEBRUARY 2011
CONCLUSIONS: GRK3 serves as a negative regulator of CXCL12/ CXCR4 in breast cancer, and the CXCR4:GRK3 expression ratio may have prognostic significance for the metastatic potential of breast cancer.
889
The Adaptive Immune Response in Cutaneous MethicillinResistant Staphylococcus Aureus Infections I. A. Myles, C. Fowler, P. Valdez, S. Datta; The National Institutes of Health, Bethesda, MD. RATIONALE: Cutaneous methicillin-resistant staphylococcus aureus (MRSA) infection is an increasingly prevalent clinical dilemma. While it is known that innate immunity and antimicrobial peptides are central in the response to MRSA skin infections, far less is known about the role of adaptive immunity. METHODS: To assess the effect of adaptive immunity we compared lesion size groups of C57BL/6 mice with: 1) two intradermal MRSA inoculations, 2) injection with diluent followed by MRSA inoculation, and 3) use of a CD4 depleting antibody prior to sequential MRSA inoculations, with each group’s inoculations spaced 21-28 days apart. Blood was periodically assessed for anti-MRSA antibodies. On day 27-34 splenocytes and draining lymph nodes were harvested and staphylococcus-specific cytokines and VBeta usage were evaluated. RESULTS: We found that neither prior MRSA exposure nor CD4 depletion had any impact on lesion area. There was no shift in VBeta usage with either singular or sequential infections. There was a significant increase in total anti-MRSA IgG, IgG1, IgG2c, and IgAwith repeat infection. CD4 depletion greatly reduced total IgG and IgG2c but not IgG1 or IgA. We found no measurable IgE. For cytokines, only IL-17 in the draining lymph nodes was significantly higher with repeat exposure. Several other cytokines were independent of exposure history, whereas splenic IL-13 was higher with initial infection. CONCLUSIONS: These results indicate that while an adaptive response is seen with MRSA infections, antibody titers, CD4 status, and T cell cytokine production do not correlate with infection outcome. (Further work on mRNA expression and histology is ongoing.)
890
Response to Cutaneous Immunization with Low Molecular Weight Subunit Keyhole Limpet Hemocyanin (KLH) H. Milgrom1, K. L. Kesler2, M. Byron2, R. J. Harbeck1, R. Holliday2, D. Y. Leung1; 1National Jewish Health, Denver, CO, 2Rho Inc, Chapel Hill, NC. RATIONALE: This study was carried out to determine whether humoral and cellular immunological responses would be provoked by cutaneous immunization with KLH. METHODS: This was an unblinded, single-center, 8-week pilot study in healthy, nonatopic young adults. Twenty-five subjects were randomized; 24 completed the study. Subjects were assigned to 4 groups. Subjects were immunized twice, 3 weeks apart by 1 of 2 routes, scarification (SS) or intradermally (ID), with 1 of 2 doses, 50 mg (3 jabs/1 loaded bifurcated needle) or 250 mg (15 jabs/ 5 needles) for SS and 100 mg or 250 mg for the ID route. Immunoglobulins were measured at baseline and 3 weeks after each immunization. DTH testing was performed at baseline and 3 weeks after the second immunization. RESULTS: Immune responses were detected in all 4 study groups (SS low, SS high, ID low, ID high). Anti-KLH IgG, IgA, and IgM production and the number of subjects responding were greater for high dose ID than for low dose ID. Results for anti-KLH IgG1-3 showed responses to both ID and SS. One subject in each treatment group showed a positive DTH result. The percentages of subjects who doubled their antibody were similar across the 4 study groups. CONCLUSIONS: We show here that it is possible to induce both humoral and cellular immune responses in healthy subjects by either ID or SS administration of KLH. This approach may be useful in studying mechanisms of immune responses in allergic skin diseases such as atopic dermatitis.