PubMed · 8948775
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L E Muñoz Espinosa. [Delta virus].. https://pubmed.ncbi.nlm.nih.gov/8948775/
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The antiapoptotic function of NF-kappaB is believed to be mediated through the induction of antiapoptotic genes. Among the antiapoptotic genes, cellular inhibitor of apoptosis protein 2 (c-IAP2/HIAP-1/MIHC) is originally identified as a molecule recruited to the tumor necrosis factor (TNF) receptor complex, and its expression is preferentially up-regulated by TNF and other stimuli activating NF-kappaB. However, direct evidence of transcriptional regulation of NF-kappaB on the c-IAP2 gene is still missing. Here, we have cloned and characterized the promoter region required for NF-kappaB-dependent transcription of the c-IAP2 gene. Sequencing of a 3.5-kilobase fragment of the 5'-flanking region of the c-IAP2 gene has identified a TATA-like sequence and potential binding sites for nuclear factor of activated T cells, interferon regulatory factor 1, activator protein 1, glucocorticoid response element, and three putative NF-kappaB binding elements. Deletion and mutational analysis of the 5'-flanking region linked to the luciferase gene revealed that transcriptional activation by TNF or interleukin 1 is mediated cooperatively by two NF-kappaB binding sites. Electrophoretic mobility shift assays characterized that the two NF-kappaB sites can be recognized and bound by the NF-kappaB p50/p65 heterodimer. In addition, the transcription of c-IAP2 promoter was strongly up-regulated when CD40 or Epstein-Barr virus latent membrane protein 1 was overexpressed.
BACKGROUND: The analysis of cytokine production is a valuable component of studies of immune response to stimulation such as pathogens, vaccines, and other immunological challenges. One highly sensitive method of cytokine evaluation involves three-color flow cytometric analysis of cytokine production in individual CD4(+) T cells. METHODS: We present four methods to enhance the acquisition and analysis of cells secreting the cytokines interferon gamma (IFNgamma), tumor necrosis factor alpha (TNFalpha), interleukin-2 (IL-2), and interleukin-4 (IL-4). Using cytomegalovirus (CMV) as the antigenic model, titration and kinetic experiments were carried out in whole blood from CMV-seropositive donors. RESULTS: CMV is most effective as a stimulating antigen when used at a dose of 5 microg/ml and for a period of at least 6 h, the first 2 h in the absence of 10 microg/ml Brefeldin A. This period of incubation can be made more convenient by the use of a "timed cooling" device, whereby the samples are automatically cooled and held at 4 degrees C at the end of incubation. Such timed cooling does not affect backgrounds or the proportion of responding cells. For certain samples, a high background can be reduced by adding fourth-color reagents. They identify and allow for elimination of monocytes and activated platelets, which contribute to false positive staining. CONCLUSIONS: These optimizations make the assay both convenient for use in whole blood samples and highly reproducible (intra-assay variability is less than 10%; interassay variability is less than 25%).
OBJECTIVE: Researchers for this project evaluated compliance with the sequential poliovirus immunization schedule that uses inactivated poliovirus vaccine (IPV) for the first 2 doses of the polio immunization series, and assessed immunization coverage rates before and after implementation of this schedule at 6 public health clinics serving 1 county in Georgia. DESIGN: Immunization histories for 3 birth cohorts of infants were compared: (1) the baseline cohort, born January 1 through June 30, 1995; (2) the evaluation cohort, born January 1 through June 30, 1997, after implementation of the schedule change; and (3) the dose-3 cohort, born August 1 through November 30, 1996 (i.e., old enough to be eligible for a third dose of poliovirus vaccine following implementation of the sequential schedule). RESULTS: Following implementation of the new poliovirus immunization recommendations, 94% (534 of 567) of infants who received their first dose of poliovirus vaccine by age 3 months received IPV. Among these infants, 99.6% (532 of 534) were also up to date (UTD) for first doses of diphtheria and tetanus toxoids and acellular pertussis vaccine (DTP1/DTaP1), 99.6% (532 of 534) were UTD for first doses of hemophilus influenza type b (Hib 1), and 98.6% (527 of 534) had received at least one dose of Hepatitis B. Among infants visiting the clinics for their first or second dose of poliovirus vaccine, DTaP/DTP, and/or Hib, 76% received 3 or 4 simultaneous injections. In the dose-3 cohort, 78% (145 of 185) of infants who received a third dose of poliovirus vaccine had received 2 doses of IPV and 1 dose of oral poliovirus vaccine. CONCLUSIONS: Compliance with the recommended use of IPV for the first 2 poliovirus immunization doses as part of the sequential schedule was very high in this low-income and ethnically diverse population. Furthermore, the need for additional injections did not impede the delivery of recommended childhood immunizations.