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Diagnostic value of CD14+ CD16+ monocytes in neonatal sepsis
S220 Abstracts
Diagnostic Value of CD14+ CD16+ Monocytes in Neonatal Sepsis Y. M. E. El-Gamal1, N. M. A. Heshmat1, A. E. A. Shehab2, A. F. M. Hasaneen3; 1Pediatrics, Ain Shams University, Faculty of Medicine, Cairo, EGYPT, 2Clinical Pathology, Ain Shams University, Faculty of Medicine, Cairo, EGYPT, 3Benha Children Hospital, Benha, EGYPT. RATIONALE: CD14+CD16+ monocytes (MO) are a potent phagocytosing and antigen-presenting monocyte subpopulation. Our purpose was to examine CD14+ CD16+ MO as a possible marker for diagnosis of neonatal sepsis. METHODS: This study comprised 45 preterm neonates (1-3 days old) with a mean gestational age of 34.5 ± 1.03 weeks .They were classified into 3 groups; proven sepsis, possible sepsis and controls (n=15 for each). Laboratory investigations included; complete blood count, blood culture and sensitivity, C-reactive protein (CRP), CD14 and CD16 expression on monocytes by flow cytometry. RESULTS: The proportion of CD14+ CD16+ MO within all circulating monocytes was significantly higher in proven sepsis (75.2±13.1%) than controls (3.86±2.53%) (p<0.001). It was higher in proven than possible sepsis ( p<0.05) . There was a positive correlation between mean fluorescence intensity (MFI) of CD16+ MO and CRP ( p<0.01) and a negative correlation between it and the platelet count ( p<0.05). Neonates with possible sepsis were followed up for 48 hours,where 9 of them developed evident sepsis and showed a significant increase in MFI of CD16 expression on monocytes (p<0.01). The sensitivity and negative predictive value of CD14+ CD16+ MO% was higher than that of CRP, their specificity and positive predictive value were comparable. CONCLUSIONS: The measurement of the percentage of CD14+ CD16+ MO among circulating MO is a promising rapid and sensitive test for early diagnosis of neonatal sepsis and exclusion of infection in neonates with high risk to develop sepsis. NICU costs as well as unnecessary antibiotic use can be thus reduced.
874
Mechanisms of Isotype Determination During Immunoglobulin Class Switch Recombination in Human B-cells D. J. Fear1, N. McCloskey1, G. Felsenfeld2, H. Gould1; 1Randall Division, King’s College London, London, UNITED KINGDOM, 2National Institutes of Health, Bethesda, MD. RATIONALE: To investigate the mechanisms regulating isotype determination during immunoglobulin class switch recombination (CSR). METHODS: We have used quantitative RT-PCR and single cell RT-PCR analysis to measure the levels of immunoglobulin germline gene transcripts (GLT) in human B-cells upon IL-4 stimulation. The role GLT play in isotype determination is followed by analysing the outcome of CSR in these cells. Potential mechanisms of isotype determination have been investigated by mapping DNA CpG methylation over the Ig locus. RESULTS: We have demonstrated that a significant proportion (>40%) of unstimulated naïve B-cells constitutively express GLT from multiple immunoglobulin isotypes. However, IL-4 greatly increases the levels of germline gene transcription prior to CSR. This is brought about by an increase in the levels of germline gene transcripts being produced by these cells, rather than increasing the proportion of cells producing GLT de novo. However, we see no change in the general pattern of CpG methylation after IL-4 stimulation. CONCLUSIONS: We conclude that the entire immunoglobulin heavy chain locus is in an accessible conformation prior to cytokine stimulation; thus chromatin “opening” does not appear to play a role in isotype determination by CSR. Rather, CSR appears to be regulated by the level of germline gene transcription occurring at different immunoglobulin loci. Although we were unable to detect any differences in the patterns of CpG methylation after IL-4 stimulation, we cannot as yet exclude the possibility that these changes only occur in the small proportion of cells that undergo CSR to a particular immunoglobulin gene. Funding: Medical Research Council
875
J ALLERGY CLIN IMMUNOL FEBRUARY 2005
876
Activated T Cells Secrete Histamine Releasing Factor
H. Lu, Y. Yang, D. P. Huston, X. Yang; Dept. of Medicine, Baylor College of Medicine, Houston, TX. RATIONALE: Histamine is a major mediator of atopy and asthma. Histamine release from mast cells and basophils can be induced by a variety of stimuli, including histamine releasing factor (HRF). HRF is ubiquitously expressed as an intracellular protein and was previously shown to be secreted by monocytes and macrophages. Given the central role that T cells play in regulating allergic inflammation, we hypothesized that activated T cells can secret HRF. METHODS: Jurkat T cells were stimulated with phorbol 12-myristate acetate (PMA) and calcium ionophore (I). HRF in supernatant and cells was analyzed by Western blot. HRF secretory pathways were investigated using pharmacological inhibitors. RESULTS: At baseline, HRF was present intracellularly but not in supernatant. PMA/I activation resulted in HRF in supernatant at 24 hours but not at 6 hours. HRF secretion was not inhibited by the classical secretory pathway inhibitors Brefeldin and monesin, indicating that HRF was secreted from T cells via a non-classical secretory pathway, similar to that demonstrated for interleukin-1. HRF secretion was regulated by the PI3 kinase pathway, as judged by inhibition of HRF secretion by a PI3 kinase inhibitor. CONCLUSIONS: This is the first demonstration that T cells can express and secret HRF. Secretion of HRF is dependent on PMA/I activation signals, occurs after 6 hrs of stimulation, and utilizes the non-classical secretory pathway. These studies implicate T cells as a potentially important source of HRF in allergic inflammation.
TUESDAY
Histamine and Its Antagonist (Desloratadine) Influence Apoptosis of CD4+CD25+ Cells in Atopic Patients and Healthy Individuals I. Kaidashev1, N. Kutsenko1, O. Borzih1, L. Mular1, L. M. DuBuske2; 1Ukrainian Medical Stomatological Academy, Poltava, UKRAINE, 2Immunology Research Institute of New England, Gardner, MA. RATIONALE: The influence of desloratadine(D) and histamine(H) on apoptosis of CD4+CD25+ regulatory T cells in atopic patients and healthy individuals was assessed. METHODS: Patients (n=6) with positive skin prick tests (SPT) to house dust mite allergens and positive clinical history were treated with D 5mg once daily for 10 days. The control group was 6 individuals without symptoms and with negative SPT. Histamine was added to cultures of Ficoll density gradient purified peripheral blood mononuclear cells (PBMC) from the control group in doses of 0.01, 0.1 and 1 M, or D was added in doses of 0.1, 1 and 10 M, and incubated up to 96 hours. Lymphocyte subpopulations and apoptosis were analyzed by flow cytometry. RESULTS: After treatment with D allergic patients had no significant changes in number of CD4+CD25+ cells, but co-culturing PBMC from patients treated with D with specific allergen increased the CD4+CD25+ cells. Treatment with D led to a significant increase in expression of the antiapoptotic protein bcl-2 in CD4 + cells, and a decrease in CD95 expression on CD4+ cells, in addition to a decrease in Treg cells in the late stage of apoptosis (AnV+PI+ cells). In vitro H induced apoptosis of CD4+CD25+ T cells in healthy individuals, while D inhibited apoptosis. CONCLUSIONS: Desloratadine prevents allergen-specific apoptosis of CD4+CD25+ T-regulatory cells in patients with allergic disease and spontaneous apoptosis in healthy individuals. Histamine induces apoptosis of CD4+CD25+ T cells in cultures of PBMC from healthy individuals. The type 1 histamine receptor may assist regulation of apoptosis of CD4+CD25+ T cells. Funding: Ukrainian Medical Stomatological Academy
877
Diagnostic Value of CD14+ CD16+ Monocytes in Neonatal Sepsis Y. M. E. El-Gamal1, N. M. A. Heshmat1, A. E. A. Shehab2, A. F. M. Hasaneen3; 1Pediatrics, Ain Shams University, Faculty of Medicine, Cairo, EGYPT, 2Clinical Pathology, Ain Shams University, Faculty of Medicine, Cairo, EGYPT, 3Benha Children Hospital, Benha, EGYPT. RATIONALE: CD14+CD16+ monocytes (MO) are a potent phagocytosing and antigen-presenting monocyte subpopulation. Our purpose was to examine CD14+ CD16+ MO as a possible marker for diagnosis of neonatal sepsis. METHODS: This study comprised 45 preterm neonates (1-3 days old) with a mean gestational age of 34.5 ± 1.03 weeks .They were classified into 3 groups; proven sepsis, possible sepsis and controls (n=15 for each). Laboratory investigations included; complete blood count, blood culture and sensitivity, C-reactive protein (CRP), CD14 and CD16 expression on monocytes by flow cytometry. RESULTS: The proportion of CD14+ CD16+ MO within all circulating monocytes was significantly higher in proven sepsis (75.2±13.1%) than controls (3.86±2.53%) (p<0.001). It was higher in proven than possible sepsis ( p<0.05) . There was a positive correlation between mean fluorescence intensity (MFI) of CD16+ MO and CRP ( p<0.01) and a negative correlation between it and the platelet count ( p<0.05). Neonates with possible sepsis were followed up for 48 hours,where 9 of them developed evident sepsis and showed a significant increase in MFI of CD16 expression on monocytes (p<0.01). The sensitivity and negative predictive value of CD14+ CD16+ MO% was higher than that of CRP, their specificity and positive predictive value were comparable. CONCLUSIONS: The measurement of the percentage of CD14+ CD16+ MO among circulating MO is a promising rapid and sensitive test for early diagnosis of neonatal sepsis and exclusion of infection in neonates with high risk to develop sepsis. NICU costs as well as unnecessary antibiotic use can be thus reduced.
874
Mechanisms of Isotype Determination During Immunoglobulin Class Switch Recombination in Human B-cells D. J. Fear1, N. McCloskey1, G. Felsenfeld2, H. Gould1; 1Randall Division, King’s College London, London, UNITED KINGDOM, 2National Institutes of Health, Bethesda, MD. RATIONALE: To investigate the mechanisms regulating isotype determination during immunoglobulin class switch recombination (CSR). METHODS: We have used quantitative RT-PCR and single cell RT-PCR analysis to measure the levels of immunoglobulin germline gene transcripts (GLT) in human B-cells upon IL-4 stimulation. The role GLT play in isotype determination is followed by analysing the outcome of CSR in these cells. Potential mechanisms of isotype determination have been investigated by mapping DNA CpG methylation over the Ig locus. RESULTS: We have demonstrated that a significant proportion (>40%) of unstimulated naïve B-cells constitutively express GLT from multiple immunoglobulin isotypes. However, IL-4 greatly increases the levels of germline gene transcription prior to CSR. This is brought about by an increase in the levels of germline gene transcripts being produced by these cells, rather than increasing the proportion of cells producing GLT de novo. However, we see no change in the general pattern of CpG methylation after IL-4 stimulation. CONCLUSIONS: We conclude that the entire immunoglobulin heavy chain locus is in an accessible conformation prior to cytokine stimulation; thus chromatin “opening” does not appear to play a role in isotype determination by CSR. Rather, CSR appears to be regulated by the level of germline gene transcription occurring at different immunoglobulin loci. Although we were unable to detect any differences in the patterns of CpG methylation after IL-4 stimulation, we cannot as yet exclude the possibility that these changes only occur in the small proportion of cells that undergo CSR to a particular immunoglobulin gene. Funding: Medical Research Council
875
J ALLERGY CLIN IMMUNOL FEBRUARY 2005
876
Activated T Cells Secrete Histamine Releasing Factor
H. Lu, Y. Yang, D. P. Huston, X. Yang; Dept. of Medicine, Baylor College of Medicine, Houston, TX. RATIONALE: Histamine is a major mediator of atopy and asthma. Histamine release from mast cells and basophils can be induced by a variety of stimuli, including histamine releasing factor (HRF). HRF is ubiquitously expressed as an intracellular protein and was previously shown to be secreted by monocytes and macrophages. Given the central role that T cells play in regulating allergic inflammation, we hypothesized that activated T cells can secret HRF. METHODS: Jurkat T cells were stimulated with phorbol 12-myristate acetate (PMA) and calcium ionophore (I). HRF in supernatant and cells was analyzed by Western blot. HRF secretory pathways were investigated using pharmacological inhibitors. RESULTS: At baseline, HRF was present intracellularly but not in supernatant. PMA/I activation resulted in HRF in supernatant at 24 hours but not at 6 hours. HRF secretion was not inhibited by the classical secretory pathway inhibitors Brefeldin and monesin, indicating that HRF was secreted from T cells via a non-classical secretory pathway, similar to that demonstrated for interleukin-1. HRF secretion was regulated by the PI3 kinase pathway, as judged by inhibition of HRF secretion by a PI3 kinase inhibitor. CONCLUSIONS: This is the first demonstration that T cells can express and secret HRF. Secretion of HRF is dependent on PMA/I activation signals, occurs after 6 hrs of stimulation, and utilizes the non-classical secretory pathway. These studies implicate T cells as a potentially important source of HRF in allergic inflammation.
TUESDAY
Histamine and Its Antagonist (Desloratadine) Influence Apoptosis of CD4+CD25+ Cells in Atopic Patients and Healthy Individuals I. Kaidashev1, N. Kutsenko1, O. Borzih1, L. Mular1, L. M. DuBuske2; 1Ukrainian Medical Stomatological Academy, Poltava, UKRAINE, 2Immunology Research Institute of New England, Gardner, MA. RATIONALE: The influence of desloratadine(D) and histamine(H) on apoptosis of CD4+CD25+ regulatory T cells in atopic patients and healthy individuals was assessed. METHODS: Patients (n=6) with positive skin prick tests (SPT) to house dust mite allergens and positive clinical history were treated with D 5mg once daily for 10 days. The control group was 6 individuals without symptoms and with negative SPT. Histamine was added to cultures of Ficoll density gradient purified peripheral blood mononuclear cells (PBMC) from the control group in doses of 0.01, 0.1 and 1 M, or D was added in doses of 0.1, 1 and 10 M, and incubated up to 96 hours. Lymphocyte subpopulations and apoptosis were analyzed by flow cytometry. RESULTS: After treatment with D allergic patients had no significant changes in number of CD4+CD25+ cells, but co-culturing PBMC from patients treated with D with specific allergen increased the CD4+CD25+ cells. Treatment with D led to a significant increase in expression of the antiapoptotic protein bcl-2 in CD4 + cells, and a decrease in CD95 expression on CD4+ cells, in addition to a decrease in Treg cells in the late stage of apoptosis (AnV+PI+ cells). In vitro H induced apoptosis of CD4+CD25+ T cells in healthy individuals, while D inhibited apoptosis. CONCLUSIONS: Desloratadine prevents allergen-specific apoptosis of CD4+CD25+ T-regulatory cells in patients with allergic disease and spontaneous apoptosis in healthy individuals. Histamine induces apoptosis of CD4+CD25+ T cells in cultures of PBMC from healthy individuals. The type 1 histamine receptor may assist regulation of apoptosis of CD4+CD25+ T cells. Funding: Ukrainian Medical Stomatological Academy
877