Potential killers exposed: tracking endogenous influenza-specific CD8+ T cells.

Current influenza A virus (IAV) vaccines stimulate antibody responses that are directed against variable regions of the virus, and are therefore ineffective against divergent strains. As CD8+ T cells target the highly conserved, internal IAV proteins, they have the potential to increase heterosubtypic immunity. Early T-cell priming events influence lasting memory, which is required for long-term protection. However, the early responding, IAV-specific cells are difficult to monitor because of their low frequencies. Here, we tracked the dissemination of endogenous IAV-specific CD8+ T cells during the initial phases of the immune response following IAV infection. We exposed a significant population of recently activated, CD25+ CD43+ IAV-specific T cells that were not detected by tetramer staining. By tracking this population, we found that initial T-cell priming occurred in the mediastinal lymph nodes, which gave rise to the most expansive IAV-specific CD8+ T-cell population. Subsequently, IAV-specific CD8+ T cells dispersed to the bronchoalveolar lavage and blood, followed by spleen and liver, and finally to the lung. These data provide important insight into the priming and tissue dispersion of an endogenous CD8+ T-cell response. Importantly, the CD25+ CD43+ phenotype identifies an inclusive population of early responding CD8+ T cells, which may provide insight into TCR repertoire selection and expansion. A better understanding of this response is critical for designing improved vaccines that target CD8+ T cells.

Efficacy of the influenza vaccine in patients with malignant lymphoma.

BACKGROUND: The benefits and efficacy of the influenza vaccine have been controversial and have had mixed reviews in the recent literature. Immunosuppressed patients and those receiving chemotherapy are particularly at risk for infectious complications and are therefore given high priority to receiving prophylactic vaccines. METHOD: We administered the influenza vaccine to 29 patients with malignant lymphoma who were receiving chemotherapy or had recently completed therapy during the flu season of 2003-2004. An aged-matched control group received the same vaccine during the same period. The ability of both groups to mount a protective titer of antibodies to the antigens in the vaccine was measured. RESULTS: Three of 29 patients (10%) in the lymphoma group were able to mount a 4-fold titer to at least one of the influenza A antigens. One patient developed a protective titer to both influenza A and B antigens and 3 of 29 responded to the influenza B antigen. In the control group 13 of 29 (45%) responded to an influenza A antigen and 14 of 29 (48%) had a 4-fold response to the B antigen. Seven of 29 controls (24%) had a 4-fold increase in their titers to both the A and B antigens. CONCLUSIONS: This study confirmed the low incidence of response or efficacy to the influenza vaccine reported in previous studies. Only a small percentage (10%) of immunosuppressed patients with malignant lymphoma responded with a 4-fold increase in their antibody titer to the major antigens of the 2003 influenza vaccine. Most interestingly, less than 50% of the aged-matched control population studied responded with a 4-fold increase in their antibody titer. Additional studies are needed to determine methods for improving the efficacy of the vaccine and the effectiveness of the influenza vaccination program in preventing influenza infections in the United States.

Identification of mosquito bloodmeal source by terminal restriction fragment length polymorphism profile analysis of the cytochrome B gene.

Previous studies have shown that polymerase chain reaction (PCR) heteroduplex analysis (HDA) of the cytochrome B (cytb) gene is useful in identifying mosquito bloodmeals derived from avian hosts. However, interpretation of PCR-HDA gels is performed visually, which can make it difficult to analyze large numbers of specimens and to compare results between laboratories. We investigated the utility of a terminal restriction fragment length polymorphism (T-RFLP) assay to analyze cytb PCR products. PCR was performed on 123 blood or tissue samples from 55 avian, 13 mammalian, and one amphibian species by using end-labeled primers to amplify a 358-bp segment of cytb. Each PCR product was sequenced to determine predicted terminal restriction fragment (TRF) profiles. Additionally, experimental TRFs were determined by sizing fragments from restriction endonuclease digests with capillary electrophoresis. A Web-based searchable database was created to compare unknown mosquito bloodmeal TRF profiles against sequence-predicted and experimentally derived terminal fragment lengths of known vertebrates. The predictive value of experimental profiles was found to be accurate to the species level for 67 of 69 species (97%). Fifty-nine field-collected mosquitoes were tested to determine the bloodmeal source using the T-RFLP method. The bloodmeal source from 50 of these mosquitoes was identified by comparing the TRF profile of the unknown source against the cytochrome B database. The bloodmeal source from the remaining nine mosquitoes was not identified as no known profile matched the experimentally derived profile. T-RFLP analysis is a highly reproducible technique and the searchable TRF database is continually being expanded to include additional species from diverse geographic areas.

Rapid molecular diagnosis of lactobacillus bacteremia by terminal restriction fragment length polymorphism analysis of the 16S rRNA gene.

OBJECTIVE: Bacteremia due to lactobacilli is uncommon, yet it is increasing in frequency, especially among immunosuppressed patients. In the clinical laboratory, lactobacilli must be subcultured from positive blood cultures before identification by traditional biochemical methods. Delays in diagnosis are significant because the organisms are inherently resistant to vancomycin, a drug frequently prescribed for empiric therapy for gram-positive bacteremia. Recently, we developed a rapid terminal-restriction fragment length polymorphism (T-RFLP) diagnostic assay based on species-specific variations in the bacterial 16S rRNA gene. We sought to apply this technique to the identification of Lactobacillus spp. from three cases of bacteremia. DESIGN: The results of the T-RFLP analysis are compared with two standard biochemical identification methods. METHODS: Lactobacillus strains were isolated from positive clinical blood cultures. Initial suspect cultures were subcultured and characterized using an automated substrate hydrolysis system and Lactobacillus carbohydrate fermentation profiles. Further biochemical and molecular analyses were performed from isolates propagated in Lactobacillus MRS broth. DNA was extracted and the 16S rRNA gene sequenced. Two sets of fluorescent labeled primers targeting the 16S rRNA gene were used for polymerase chain reaction (PCR) with chromosomal preparations from reference strains and blood isolates. The PCR products were digested with restriction enzymes and terminal-restriction fragment profile analysis performed. RESULTS: T-RFLP analysis correctly identified the Lactobacillus species in each case. T-RFLP analysis could be completed within 8 hours of obtaining a positive blood culture as compared to more than the 24 to 48 hours required for traditional culturing and biochemical characterizations. CONCLUSION: T-RFLP analysis allows for rapid identification of Lactobacillus directly from positive blood cultures and circumvents the requirement for subculture. Reduced diagnostic time has implications for duration of infection, the cost of patient care, length of hospitalization, development of broad-spectrum antibiotic resistance, and mortality due to bacteremia. T-RFLP profiling represents a highly reproducible and predictive source for identification of many organisms associated with bacteremia.