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The Role of Endotoxin and Endotoxin Immune Responses in End Stage Renal Disease and Hemodialysis
S224 Abstracts
863
Lymphoid Cell Viability Modulation by Human T-Cell Leukemia Virus Type 2 Tax Protein C. Barrios1, L. Castillo1, C. Giam2, M. Lairmore3, C. Dong1, L. Wu1, M. Beilke1; 1Medical College Of Wisconsin, Milwaukee, WI, 2Uniformed Services University of the Health Sciences, Bethesda, MD, 3The Ohio State University, Columbus, OH. RATIONALE: HIV-1 and HTLV-2 co-infections have been documented to be associated with survival benefit, delayed progression to AIDS, and delayed rates of CD41 T cell decline. The survival benefit has been linked to the upregulated expression of the HTLV-2 transcriptional activating protein, known as Tax2. We hypothesize that Tax2 protein promotes T cell survival and delayed CD4 1 T cell decline in HIV-1 and HTLV-2 co-infections through inhibition of cellular apoptosis. METHODS: Peripheral blood mononuclear cells (PBMCs) of healthy donors were cultured with recombinant Tax2 protein derived from E. coli transformed with expression constructs. Extracellular bacterial extract (ebe) was used as background control. Cell viability was determined by trypan-blue-exclusion-test, and levels of early and late apoptosis were measured by FACS analysis of the cell surface expression of translocated phosphatidylserine and 7-amino-actinomycin-D interaction with DNA of dead cells. RESULTS: The ability of the recombinant Tax2 protein to bind the Tax Response Element (TRE) and initiate transcription was established using a luciferase reporter assay. Significant increase in PBMC viability was determined in cells treated with recombinant Tax2 protein, compared to ebetreated and untreated PBMCs (p < 0.01). Tax2-treated PBMCs showed a higher percentage of live cells (81%) and lower percentage of apoptotic cells (19%) compared to the untreated PBMCs (60% live and 40% dead cells) or the ebe-treated PBMCs (70% live and 30% dead). CONCLUSIONS: Tax2 protein enhances PBMC viability and survival in vitro. These results support the contention that upregulated HTLV-2 Tax gene expression contributes to the survival benefit observed in patients with HIV-1/HTLV-2 co-infection.
864
TUESDAY
Prenatal Administration Of Lactobacillus rhamnosus GG Can Modulate Intestinal Microbiota In Infants At High Risk Of Allergic Disease M. L. K. Tang1,2, S. J. Lahtinen1,3, R. J. Boyle1,2, S. Kivivuori1,2, F. Oppedisano1, K. R. Smith1, R. M. Robins-Browne1,2, S. Salminen3; 1Murdoch Children’s Research Institute, Melbourne, Australia, 2University of Melbourne, Melbourne, Australia, 3Functional Foods Forum, University of Turku, Turku, Finland. RATIONALE: Perinatal supplementation with probiotic bacteria such as Lactobacillus rhamnosus GG (LGG) has been shown to prevent the development of allergic disease, however the mechanisms of action remain unclear. Infants who subsequently develop allergic disease have altered composition of the intestinal microbiota - in particular differences in the genus Bifidobacterium have been reported. Probiotics may modulate infant intestinal microbiota colonization, particularly Bifidobacterium colonization. We examined the effects of LGG administration to pregnant women on infant intestinal microbiota composition. METHODS: 122 women were randomised to receive LGG or placebo from 36 weeks gestation until delivery. Infants were at high risk of developing allergic disease due to a history of allergic disease in parent or sibling. The composition of infant and maternal fecal Bifidobacterium microbiota was determined by terminal restriction fragment length polymorphism at birth and during the first 90d. LGG colonization of mothers and infants was also assessed. RESULTS: At 90d, infants in the LGG group were more frequently colonized with B. longum than the placebo group (p 5 0.01; prevalence ratio 1.35, 95% CI 1.06-1.72). Compared to the placebo group, the Bifidobacterium microbiota harboured by the infants in the LGG group more closely resembled the typical Bifidobacterium microbiota of healthy breast-fed infants. No evidence was found that prenatal probiotic treatment increased infant intestinal colonization with the administered probiotic, despite increased maternal colonization.
J ALLERGY CLIN IMMUNOL FEBRUARY 2009
CONCLUSIONS: Administration of LGG to mothers during late pregnancy can influence the development of infant Bifidobacterium microbiota, and may offer a new approach to the prevention of allergic diseases.
865
The Role of Endotoxin and Endotoxin Immune Responses in End Stage Renal Disease and Hemodialysis V. A. Beloglazov1, A. V. Klimchuck1, A. I. Gordienko1, A. I. Bakova1, I. V. DuBuske2, L. M. DuBuske2; 1Crimean State Medical University, Simferopol, Ukraine, 2Immunology Research Institute of New England, Gardner, MA. RATIONALE: Endotoxin from gram-negative bacteria may have an influence on the duration and disease complications of end stage renal disease (ESRD) requiring hemodialysis (HD). Anti-endotoxin antibodies (anti-ET Ab) play a crucial role in endotoxin neutralization. Specific anti-ET responses may reflect the overall endotoxin impact in ESRD. METHODS: 79 patients with ESRD from 32 to 47 years old were assessed. Levels of IgA, IgM, IgG classes of anti-ET Ab were measured by ELISA. Blood samples were obtained twice from each patient with ESRD, before HD and 3 days after HD. Levels of similar antibodies from 18 healthy individuals were assessed as a control. RESULTS: Serum levels of anti-ET-Ig A were significantly increased by 107.5% before HD, and by 118.6% after HD (p < 0.001) compared with the normal range. Prior to HD there was marked enhancement of anti-ET IgG, being 1.8 fold greater (p < 0.001) than normal controls but after HD serum levels of anti-ET IgG were normal. Levels of anti-ET IgM in ESRD patients undergoing HD were not different from the normal controls. CONCLUSIONS: Immune responses to endotoxin in patients with ESRD suggests a possible role of gram-negative bacteria in the pathogenesis of ESRD. HD reduces serum levels anti-ET IgG perhaps related to consumption of these antibodies due enhanced translocation of ET from the GI tract and subsequent activation of inflammatory cells during HD. Strategies to limit inflammatory changes during HD may include reduction of ET translocation from the GI tract.
866
Calcitriol is a Growth Factor for Human Airway Epithelial Cells P. E. Dahlberg, R. A. Brockman-Schneider, J. E. Gern; University of Wisconsin Hospital and Clinics, Madison, WI. RATIONALE: Vitamin D, postulated to have protective effects on asthma, also promotes wound healing in keratinocytes. Effects of vitamin D on growth and development of airway epithelium have not yet been established. METHODS: Human epithelial cells obtained from healthy lung donors were grown on an air-liquid interface with or without calcitriol (0, 10, 100 mM). Transepithelial resistance was monitored. After 24 days, representative sections of the tissues were mounted and stained. Nuclei on six representative H&E stained cross-sectional samples from each treatment group were manually counted, and mean tissue thickness was determined (SigmaScan, Systat, San Jose, CA). RESULTS: Transepithelial resistance reached 1500 Ohms in the control cells after 8 days, in the 10 mM calcitriol group after 10 days, and in the 100 mM calcitriol group after 11 days. Escalating concentrations of calcitriol caused increases in cell layer thickness (control: 31 1/2 3 mm, 10 mM calcitriol: 42 1/2 3 mm, 100 mM calcitriol: 68 1/2 17 mm), nuclei per 100 mm (control: 25 1/2 1, 10 mM calcitriol: 32 1/23, 100 mM calcitriol: 36 1/2 6) and cell size (control: 125 1/2 10 mm2, 10 mM calcitriol: 133 1/27 mm2, 100 mM calcitriol: 186 1/2 18 mm2). Both calcitriol treatments were associated with the presence of an additional spindleshaped cell-layer on cross-section. CONCLUSIONS: Calcitriol in physiologic doses is a potent growth factor for cultured airway epithelial cells.
863
Lymphoid Cell Viability Modulation by Human T-Cell Leukemia Virus Type 2 Tax Protein C. Barrios1, L. Castillo1, C. Giam2, M. Lairmore3, C. Dong1, L. Wu1, M. Beilke1; 1Medical College Of Wisconsin, Milwaukee, WI, 2Uniformed Services University of the Health Sciences, Bethesda, MD, 3The Ohio State University, Columbus, OH. RATIONALE: HIV-1 and HTLV-2 co-infections have been documented to be associated with survival benefit, delayed progression to AIDS, and delayed rates of CD41 T cell decline. The survival benefit has been linked to the upregulated expression of the HTLV-2 transcriptional activating protein, known as Tax2. We hypothesize that Tax2 protein promotes T cell survival and delayed CD4 1 T cell decline in HIV-1 and HTLV-2 co-infections through inhibition of cellular apoptosis. METHODS: Peripheral blood mononuclear cells (PBMCs) of healthy donors were cultured with recombinant Tax2 protein derived from E. coli transformed with expression constructs. Extracellular bacterial extract (ebe) was used as background control. Cell viability was determined by trypan-blue-exclusion-test, and levels of early and late apoptosis were measured by FACS analysis of the cell surface expression of translocated phosphatidylserine and 7-amino-actinomycin-D interaction with DNA of dead cells. RESULTS: The ability of the recombinant Tax2 protein to bind the Tax Response Element (TRE) and initiate transcription was established using a luciferase reporter assay. Significant increase in PBMC viability was determined in cells treated with recombinant Tax2 protein, compared to ebetreated and untreated PBMCs (p < 0.01). Tax2-treated PBMCs showed a higher percentage of live cells (81%) and lower percentage of apoptotic cells (19%) compared to the untreated PBMCs (60% live and 40% dead cells) or the ebe-treated PBMCs (70% live and 30% dead). CONCLUSIONS: Tax2 protein enhances PBMC viability and survival in vitro. These results support the contention that upregulated HTLV-2 Tax gene expression contributes to the survival benefit observed in patients with HIV-1/HTLV-2 co-infection.
864
TUESDAY
Prenatal Administration Of Lactobacillus rhamnosus GG Can Modulate Intestinal Microbiota In Infants At High Risk Of Allergic Disease M. L. K. Tang1,2, S. J. Lahtinen1,3, R. J. Boyle1,2, S. Kivivuori1,2, F. Oppedisano1, K. R. Smith1, R. M. Robins-Browne1,2, S. Salminen3; 1Murdoch Children’s Research Institute, Melbourne, Australia, 2University of Melbourne, Melbourne, Australia, 3Functional Foods Forum, University of Turku, Turku, Finland. RATIONALE: Perinatal supplementation with probiotic bacteria such as Lactobacillus rhamnosus GG (LGG) has been shown to prevent the development of allergic disease, however the mechanisms of action remain unclear. Infants who subsequently develop allergic disease have altered composition of the intestinal microbiota - in particular differences in the genus Bifidobacterium have been reported. Probiotics may modulate infant intestinal microbiota colonization, particularly Bifidobacterium colonization. We examined the effects of LGG administration to pregnant women on infant intestinal microbiota composition. METHODS: 122 women were randomised to receive LGG or placebo from 36 weeks gestation until delivery. Infants were at high risk of developing allergic disease due to a history of allergic disease in parent or sibling. The composition of infant and maternal fecal Bifidobacterium microbiota was determined by terminal restriction fragment length polymorphism at birth and during the first 90d. LGG colonization of mothers and infants was also assessed. RESULTS: At 90d, infants in the LGG group were more frequently colonized with B. longum than the placebo group (p 5 0.01; prevalence ratio 1.35, 95% CI 1.06-1.72). Compared to the placebo group, the Bifidobacterium microbiota harboured by the infants in the LGG group more closely resembled the typical Bifidobacterium microbiota of healthy breast-fed infants. No evidence was found that prenatal probiotic treatment increased infant intestinal colonization with the administered probiotic, despite increased maternal colonization.
J ALLERGY CLIN IMMUNOL FEBRUARY 2009
CONCLUSIONS: Administration of LGG to mothers during late pregnancy can influence the development of infant Bifidobacterium microbiota, and may offer a new approach to the prevention of allergic diseases.
865
The Role of Endotoxin and Endotoxin Immune Responses in End Stage Renal Disease and Hemodialysis V. A. Beloglazov1, A. V. Klimchuck1, A. I. Gordienko1, A. I. Bakova1, I. V. DuBuske2, L. M. DuBuske2; 1Crimean State Medical University, Simferopol, Ukraine, 2Immunology Research Institute of New England, Gardner, MA. RATIONALE: Endotoxin from gram-negative bacteria may have an influence on the duration and disease complications of end stage renal disease (ESRD) requiring hemodialysis (HD). Anti-endotoxin antibodies (anti-ET Ab) play a crucial role in endotoxin neutralization. Specific anti-ET responses may reflect the overall endotoxin impact in ESRD. METHODS: 79 patients with ESRD from 32 to 47 years old were assessed. Levels of IgA, IgM, IgG classes of anti-ET Ab were measured by ELISA. Blood samples were obtained twice from each patient with ESRD, before HD and 3 days after HD. Levels of similar antibodies from 18 healthy individuals were assessed as a control. RESULTS: Serum levels of anti-ET-Ig A were significantly increased by 107.5% before HD, and by 118.6% after HD (p < 0.001) compared with the normal range. Prior to HD there was marked enhancement of anti-ET IgG, being 1.8 fold greater (p < 0.001) than normal controls but after HD serum levels of anti-ET IgG were normal. Levels of anti-ET IgM in ESRD patients undergoing HD were not different from the normal controls. CONCLUSIONS: Immune responses to endotoxin in patients with ESRD suggests a possible role of gram-negative bacteria in the pathogenesis of ESRD. HD reduces serum levels anti-ET IgG perhaps related to consumption of these antibodies due enhanced translocation of ET from the GI tract and subsequent activation of inflammatory cells during HD. Strategies to limit inflammatory changes during HD may include reduction of ET translocation from the GI tract.
866
Calcitriol is a Growth Factor for Human Airway Epithelial Cells P. E. Dahlberg, R. A. Brockman-Schneider, J. E. Gern; University of Wisconsin Hospital and Clinics, Madison, WI. RATIONALE: Vitamin D, postulated to have protective effects on asthma, also promotes wound healing in keratinocytes. Effects of vitamin D on growth and development of airway epithelium have not yet been established. METHODS: Human epithelial cells obtained from healthy lung donors were grown on an air-liquid interface with or without calcitriol (0, 10, 100 mM). Transepithelial resistance was monitored. After 24 days, representative sections of the tissues were mounted and stained. Nuclei on six representative H&E stained cross-sectional samples from each treatment group were manually counted, and mean tissue thickness was determined (SigmaScan, Systat, San Jose, CA). RESULTS: Transepithelial resistance reached 1500 Ohms in the control cells after 8 days, in the 10 mM calcitriol group after 10 days, and in the 100 mM calcitriol group after 11 days. Escalating concentrations of calcitriol caused increases in cell layer thickness (control: 31 1/2 3 mm, 10 mM calcitriol: 42 1/2 3 mm, 100 mM calcitriol: 68 1/2 17 mm), nuclei per 100 mm (control: 25 1/2 1, 10 mM calcitriol: 32 1/23, 100 mM calcitriol: 36 1/2 6) and cell size (control: 125 1/2 10 mm2, 10 mM calcitriol: 133 1/27 mm2, 100 mM calcitriol: 186 1/2 18 mm2). Both calcitriol treatments were associated with the presence of an additional spindleshaped cell-layer on cross-section. CONCLUSIONS: Calcitriol in physiologic doses is a potent growth factor for cultured airway epithelial cells.