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.

Paired analysis of TCRα and TCRß chains at the single-cell level in mice.

Characterizing the TCRα and TCRß chains expressed by T cells responding to a given pathogen or underlying autoimmunity helps in the development of vaccines and immunotherapies, respectively. However, our understanding of complementary TCRα and TCRß chain utilization is very limited for pathogen- and autoantigen-induced immunity. To address this problem, we have developed a multiplex nested RT-PCR method for the simultaneous amplification of transcripts encoding the TCRα and TCRß chains from single cells. This multiplex method circumvented the lack of antibodies specific for variable regions of mouse TCRα chains and the need for prior knowledge of variable region usage in the TCRß chain, resulting in a comprehensive, unbiased TCR repertoire analysis with paired coexpression of TCRα and TCRß chains with single-cell resolution. Using CD8+ CTLs specific for an influenza epitope recovered directly from the pneumonic lungs of mice, this technique determined that 25% of such effectors expressed a dominant, nonproductively rearranged Tcra transcript. T cells with these out-of-frame Tcra mRNAs also expressed an alternate, in-frame Tcra, whereas approximately 10% of T cells had 2 productive Tcra transcripts. The proportion of cells with biallelic transcription increased over the course of a response, a finding that has implications for immune memory and autoimmunity. This technique may have broad applications in mouse models of human disease.

Physiological numbers of CD4+ T cells generate weak recall responses following influenza virus challenge.

Naive and recall CD4(+) T cell responses were probed with recombinant influenza A viruses incorporating the OVA OT-II peptide. The extent of OT-II-specific CD4(+) T cell expansion was greater following primary exposure, with secondary challenge achieving no significant increase in numbers, despite higher precursor frequencies. Adoptive transfer experiments with OT-II TCR-transgenic T cells established that the predominant memory set is CD62L(hi), whereas the CD62L(lo) precursors make little contribution to the recall response. Unlike the situation described by other investigators, in which the transfer of very large numbers of in vitro-activated CD4 effectors can modify the disease process, providing CD62L(hi) or CD62L(lo) OT-II-specific T cells at physiological levels neither enhanced virus clearance nor altered clinical progression. Some confounding effects of the transgenic model were observed, with decreasing primary expansion efficiency correlating with greater numbers of transferred cells. This was associated with increased levels of mRNA for the proapoptotic molecule Bim in cells recovered following high-dose transfer. However, even with very low numbers of transferred cells, memory T cells did not expand significantly following secondary challenge. A similar result was recorded in mice primed and boosted to respond to an endogenous IA(b)-restricted epitope derived from the influenza virus hemagglutinin glycoprotein. Depletion of CD8(+) T cells during secondary challenge generated an increased accumulation of OT-II-specific T cells but only at the site of infection. Taken together, significant expansion was not a feature of these secondary influenza-specific CD4 T cell responses and the recall of memory did not enhance recovery.

The intracellular sensor NLRP3 mediates key innate and healing responses to influenza A virus via the regulation of caspase-1.

Virus-induced interlukin-1beta (IL-1beta) and IL-18 production in macrophages are mediated via caspase-1 pathway. Multiple microbial components, including viral RNA, are thought to trigger assembly of the cryopyrin inflammasome resulting in caspase-1 activation. Here, we demonstrated that Nlrp3(-/-) and Casp1(-/-) mice were more susceptible than wild-type mice after infection with a pathogenic influenza A virus. This enhanced morbidity correlated with decreased neutrophil and monocyte recruitment and reduced cytokine and chemokine production. Despite the effect on innate immunity, cryopyrin-deficiency was not associated with any obvious defect in virus control or on the later emergence of the adaptive response. Early epithelial necrosis was, however, more severe in the infected mutants, with extensive collagen deposition leading to later respiratory compromise. These findings reveal a function of the cryopyrin inflammasome in healing responses. Thus, cryopyrin and caspase-1 are central to both innate immunity and to moderating lung pathology in influenza pneumonia.

TNF/iNOS-producing dendritic cells are the necessary evil of lethal influenza virus infection.

Respiratory infection with highly pathogenic influenza A viruses is characterized by the exuberant production of cytokines and chemokines and the enhanced recruitment of innate inflammatory cells. Here, we show that challenging mice with virulent influenza A viruses, including currently circulating H5N1 strains, causes the increased selective accumulation of a particular dendritic cell subset, the tipDCs, in the pneumonic airways. These tipDCs are required for the further proliferation of influenza-specific CD8(+) T cells in the infected lung, because blocking their recruitment in CCR2(-/-) mice decreases the numbers of CD8(+) effectors and ultimately compromises virus clearance. However, diminution rather than total elimination of tipDC trafficking by treatment with the peroxisome proliferator-activated receptor-gamma agonist pioglitazone moderates the potentially lethal consequences of excessive tipDC recruitment without abrogating CD8(+) T cell expansion or compromising virus control. Targeting the tipDCs in this way thus offers possibilities for therapeutic intervention in the face of a catastrophic pandemic.

Screening monoclonal antibodies for cross-reactivity in the ferret model of influenza infection.

Influenza virus infections carry a high public health cost, and pandemics are potentially catastrophic. Though the ferret is generally regarded as the best model for human influenza, few reagents are available for the analysis of cellular immunity. We thus screened monoclonal antibodies (mAbs) made for identifying immune cells in other species to see if any were cross-reactive. Flow cytometric analysis of lymphocytes isolated from blood, spleen, and lung of normal and virus-infected ferrets indicated that several mouse mAbs bound to the corresponding antigens in ferrets. Typing bronchoalveolar lavage populations from pneumonic ferrets with mAb to human CD8 showed the massive CD8+ T cell enrichment characteristic of this infection in mice. The availability of this, and several other mAbs that showed cross-reactivity, should allow us to begin the dissection of cell-mediated immunity in the ferret, which, at least from these early results, looks similar to the situation in mice.

DLL3 as a candidate gene for vertebral malformations.

Investigations have not identified a major locus for congenital vertebral malformations. Based on observations in mice, we hypothesized that mutations in DLL3, a member of the notch-signaling pathway, might contribute to human vertebral malformations. We sequenced the DLL3 gene in 50 patients with congenital vertebral malformations. A Caucasian male patient with VACTERL manifestations including a T5-T6 block vertebrae was heterozygous for a "G" to "A" missense mutation changing glycine to arginine at codon 269. This residue is conserved in mammals, including chimpanzee, mouse, dog, and rat. Additional testing in the patient did not show evidence of chromosome abnormalities. The patient's asymptomatic mother was also heterozygous for the missense mutation. Since this mutation was not observed in a control population and leads to an amino acid change, it may be clinically significant. The mutation was not found in a control population of 87 anonymous individuals. Several established mechanisms could explain the mutation in both the patient and his asymptomatic mother (susceptibility allele requiring additional environmental factors, somatic mosaicism, multigenic inheritance). Documenting the absence of the mutation in a larger control population or the presence of the mutation in additional affected patients, or documenting a functional difference in DLL3 would provide further evidence supporting its causal role.

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.

Congenital and idiopathic scoliosis: clinical and genetic aspects.

OBJECTIVE: Genetic and environmental factors influencing spinal development in lower vertebrates are likely to play a role in the abnormalities associated with human congenital scoliosis (CS) and idiopathic scoliosis (IS). An overview of the molecular embryology of spinal development and the clinical and genetic aspects of CS and IS are presented. Utilizing synteny analysis of the mouse and human genetic databases, likely candidate genes for human CS and IS were identified. DESIGN: Review and synteny analysis. METHODS: A search of the Mouse Genome Database was performed for "genes," "markers" and "phenotypes" in the categories Neurological and neuromuscular, Skeleton, and Tail and other appendages. The Online Mendelian Inheritance in Man was used to determine whether each mouse locus had a known human homologue. If so, the human homologue was assigned candidate gene status. Linkage maps of the chromosomes carrying loci with possibly relevant phenotypes, but without known human homologues, were examined and regions of documented synteny between the mouse and human genomes were identified. RESULTS: Searching the Mouse Genome Database by phenotypic category yielded 100 mutants of which 66 had been mapped. The descriptions of each of these 66 loci were retrieved to determine which among these included phenotypes of scoliosis, kinky or bent tails, other vertebral abnormalities, or disturbances of axial skeletal development. Forty-five loci of interest remained, and for 27 of these the comparative linkage maps of mouse and human were used to identify human syntenic regions to which plausible candidate genes had been mapped. CONCLUSION: Synteny analysis of mouse candidate genes for CS and IS holds promise due to the close evolutionary relationship between mice and human beings. With the identification of additional genes in animal model systems that contribute to different stages of spine development, the list of candidate genes for CS and IS will continue to grow.

Current diagnosis and treatment of infective endocarditis.

The incidence of infective endocarditis continues to rise with a yearly incidence of around 15,000 to 20,000 new cases in the USA. As a result, rapid diagnosis, effective treatment and prompt recognition of complications are essential to desirable clinical outcomes. Recent guidelines such as the Duke criteria have incorporated echocardiography for diagnosis of infective endocarditis, making this diagnostic test mandatory for patients with suspected infective endocarditis. The diversity of pathogens that can cause infective endocarditis, some of which cannot be cultured easily, makes diagnosis even more difficult. Coagulase-negative staphylococci and viridans streptococci groups continue to be the major causative microorganisms of infective endocarditis. In the case of culture-negative endocarditis or infective endocarditis caused by fastidious microorganisms, the polymerase chain reaction and probe-based diagnostic methods are available to clinical reference laboratories.