Influenza A virus-induced apoptosis is a multifactorial process: exploiting reverse genetics to elucidate the role of influenza A virus proteins in virus-induced apoptosis

Three influenza viruses, A/Puerto Rico/8/34-A/England/939/69 clone 7a (H3N2), A/Fiji/15899/83 (H1N1), and A/Victoria/3/75 (H3N2), induce different levels of apoptosis in vitro at equal moi; Clone 7a > A/Victoria > A/Fiji. Previous studies have shown that several viral proteins from clone 7a and A/Fiji, including PB2, NA, NS1, M1, and M2, induce apoptosis when expressed individually fused to the herpes simplex virus tegument protein, VP22. However, this did not reflect viral protein-protein-RNA interactions known to occur within infected cells. To explore the role of viral proteins in apoptosis under infection conditions, recombinant viruses with single or triple gene exchanges were generated using A/Victoria or clone 7a as the background virus. Inserting the A/Fiji NS or PB2 gene into A/Victoria or clone 7a significantly reduced the level of apoptosis compared to the parent virus while clone 7a PA or NP genes increased apoptosis. Inserting A/Fiji NA or HA or clone 7a NS, M, NA, or HA genes individually into A/Victoria had no significant effect on apoptosis. Surprisingly, inserting the M, NA, and HA genes of A/Fiji together into clone 7a reduced apoptosis, whereas inserting clone 7a M, NA, and HA together into A/Fiji increased apoptosis. These results suggest that no single virus protein induces apoptosis and that the combination of genes required may be strain specific, highlighting the difficulty of predicting the virulence of new strains that arise in nature. No support for the view that apoptosis is essential for high virus yields was obtained as high virus yields were obtained with viruses that induced both high and low levels of apoptosis.     

Telepathology for routine light microscopic and frozen section diagnosis

Telepathology (TP) uses telecommunication linkages to electronically capture, store, retrieve, and transmit images to distant sites. We assessed the feasibility of a dynamic real-time TP system for light microscopic (LM) diagnosis of anatomic pathology specimens, including frozen sections. Six pathologists, in 2 separate periods, read a set of 160 retrospectively retrieved slides (80 of which were frozen sections) by TP and LM. Reading times were recorded. Diagnoses were compared with the reference diagnosis (established by a group of 5 independent pathologists) and graded on a scale of 0 to 2 (2, correct; 1, incorrect but no clinical impact; 0, incorrect with clinical impact). Overall, LM was more accurate than TP compared with the reference diagnosis (score, 1.68 vs 1.54). There was no difference in accuracy between frozen section and paraffin-embedded tissue. Intraobserver agreement ranged from 82.5% to 88.2%. The average reading time was 6.0 minutes for TP and 1.4 minutes for LM. During the study, reading time decreased for TP but not for LM. These results show that despite marginally lower accuracy and longer reading times, TP isfeasible for routine light microscopic diagnosis, including frozen sections.     

Suppression of the paternal IgA-f and IgA-g allotypes in heterozygous rabbits suppressed for the paternal IgM allotype

The injection of neonatal rabbits of genotype a¹n^{8 1}g^{7 4}/a²n^{8 2}f⁷¹g⁷⁵with anti-n81 antiserum suppressess the expression of the paternal f73 and g74 IgA allotypes; the expression of the maternal 171 IgA allotype is enhanced but the expression of the g75 allotype is not significantly enhanced. The concentrations of these allotypes in serum correlated with the immunofluorescent analyses of cellular ratios in gut sections. Allotype suppression of IgA with an anti-IgM reagent supports the concept that the IgM bearing cells are the precursors of the IgA secreting cells.     

B-cell activation and allosensitization after left ventricular assist device implantation is due to T-cell activation and CD40 ligand expression

Left ventricular assist device (LVAD) implantation is frequently complicated by B-cell activation and allosensitization, posing a significant risk to successful transplant outcome. This study investigated whether B-cell hyperreactivity and alloantibody production in LVAD recipients involves T-cell dependent pathways. T-cell calcium flux and nuclear translocation of NFATc were used to determine states of T-cell activation. Flow cytometry was used to assess human T- and B-cell activation after culture with LVAD-derived biomaterial particles. Sera from LVAD recipients and controls were tested for the presence of anti-HLA antibodies, and for soluble CD40 ligand. LVAD-derived biomaterial induced rapid and sustained calcium flux into normal T cells, resulting in calcineurin-dependent nuclear translocation of NFATc. This resulted in increased T-cell expression of CD40 ligand and subsequent B-cell activation, which was reduced by inhibitors of T-cell activation (CsA or anti-CD25 mAb) or by anti-CD40 ligand mAb. LVAD recipients demonstrated higher frequencies of anti-HLA antibodies and serum levels of soluble CD40 ligand compared with heart failure controls. The results indicate that exposure of human mononuclear cells to LVAD-derived biomaterial leads to T-cell dependent B-cell activation via CD40--CD40 ligand interaction, and suggest that treatment with calcineurin inhibitors or monoclonal antibodies against either CD25 or CD40 ligand could be effective at preventing B-cell hyperreactivity and allosensitization after LVAD implantation.     

Effects of dexamethasone and transient malnutrition on rabbits infected with aerosolized Mycobacterium tuberculosis CDC1551

Malnutrition is common in the developing world, where tuberculosis is often endemic. Rabbits infected with aerosolized Mycobacterium tuberculosis that subsequently became inadvertently and transiently malnourished had compromised cell-mediated immunity comparable to that of the rabbits immunosuppressed with dexamethasone. They had significant leukopenia and reduced delayed-type hypersensitivity responses. Malnutrition dampened cell-mediated immunity and would interfere with diagnostic tests that rely on intact CD4 T-cell responses.     

Differential roles of CCL2 and CCR2 in host defense to coronavirus infection

The CC chemokine ligand 2 (CCL2, monocyte chemoattractant protein-1) is important in coordinating the immune response following microbial infection by regulating T cell polarization as well as leukocyte migration and accumulation within infected tissues. The present study examines the consequences of mouse hepatitis virus (MHV) infection in mice lacking CCL2 (CCL2(-/-)) in order to determine if signaling by this chemokine is relevant in host defense. Intracerebral infection of CCL2(-/-) mice with MHV did not result in increased morbidity or mortality as compared to either wild type or CCR2(-/-) mice and CCL2(-/-) mice cleared replicating virus from the brain. In contrast, CCR2(-/-) mice displayed an impaired ability to clear virus from the brain that was accompanied by a reduction in the numbers of antigen-specific T cells as compared to both CCL2(-/-) and wild-type mice. The paucity in T cell accumulation within the central nervous system (CNS) of MHV-infected CCR2(-/-) mice was not the result of either a deficiency in antigen-presenting cell (APC) accumulation within draining cervical lymph nodes (CLN) or the generation of virus-specific T cells within this compartment. A similar reduction in macrophage infiltration into the CNS was observed in both CCL2(-/-) and CCR2(-/-) mice when compared to wild-type mice, indicating that both CCL2 and CC chemokine receptor 2 (CCR2) contribute to macrophage migration and accumulation within the CNS following MHV infection. Together, these data demonstrate that CCR2, but not CCL2, is important in host defense following viral infection of the CNS, and CCR2 ligand(s), other than CCL2, participates in generating a protective response.     

Health-related quality-of-life measures for children with asthma: reliability and validity of the Children's Health Survey for Asthma and the Pediatric Quality of Life Inventory 3.0 Asthma Module.

BACKGROUND: Economically disadvantaged African American youth are especially vulnerable to the effects of pediatric asthma and are at increased risk for difficulties in daily functioning. Measures of health-related quality of life (HRQoL) yield important information regarding the impact of pediatric chronic illness on daily functioning. It is essential to develop and validate measures of HRQoL to detect the impact of asthma on this vulnerable population. OBJECTIVE: To examine the psychometric properties of 2 asthma-specific measures of pediatric HRQoL in a sample of economically disadvantaged African American children diagnosed as having asthma. METHODS: One hundred twenty-seven caregivers completed questionnaires regarding their child's HRQoL, asthma symptoms, health care utilization, and school absences and regarding caregiver emotional distress. The severity of the child's asthma was measured via spirometry. RESULTS: The Children's Health Survey for Asthma and the Pediatric Quality of Life Inventory 3.0 Asthma Module demonstrated adequate internal consistency reliability and validity for the present sample. Lower HRQoL was associated with poorer adherence and more health care utilization, asthma symptom days, school absences, and caregiver distress. Only the Children's Health Survey for Asthma was significantly associated with severity, when defined as airway obstruction. CONCLUSIONS: This study supports the psychometric equivalence of 2 condition-specific measures of HRQoL in a population at high risk for asthma and asthma-related problems. The utility of each measure will depend on the needs of the researcher or physician. Both measures can inform the treatment course, help identify and address barriers to treatment adherence, and inform treatment interventions.     

The development of synaptic plasticity induction rules and the requirement for postsynaptic spikes in rat hippocampal CA1 pyramidal neurones

Coincident pre- and postsynaptic activity induces synaptic plasticity at the Schaffer collateral synapse onto CA1 pyramidal neurones. The precise timing, frequency and number of coincident action potentials required to induce synaptic plasticity is currently unknown. In this study we show that the postsynaptic activity required for the induction of long-term potentiation (LTP) changes with development. In acute slices from adult rats, coincident pre- and postsynaptic theta burst stimulation (TBS) induced LTP and we show that multiple high-frequency postsynaptic spikes are required. In contrast, in acute slices from juvenile (P14) rats, TBS failed to induce LTP unless the excitatory postsynaptic potentials (EPSPs) were of sufficient magnitude to initiate action potentials. We also show that coincident individual pre- and postsynaptic action potentials are only capable of inducing LTP in the juvenile when given at a frequency greater than 5 Hz and that the timing of individual pre- and postsynaptic action potentials relative to one another is not important. Finally, we show that local tetrodotoxin (TTX) application to the soma blocked LTP in adults, but not juveniles. These data demonstrate that somatic spiking is more important for LTP induction in the adult as opposed to juvenile rats and we hypothesize that the basis for this is the ability of action potentials in the postsynaptic CA1 pyramidal neurone to back-propagate into the dendrites. Therefore, the pre- and postsynaptic activity patterns required to induce LTP mature as the hippocampus develops.     

Variable clinical presentation of lysosomal beta-mannosidosis in patients with null mutations

Beta-mannosidosis is an autosomal recessive lysosomal storage disease resulting from a deficiency of the lysosomal enzyme beta-mannosidase. The clinical manifestations of this disease in reported human cases are very heterogeneous ranging from relatively mild to moderately severe. This is in contrast with the severe prenatal onset seen in ruminant beta-mannosidosis. In humans, mental retardation, hearing loss, frequent infections, and behavioral problems are relatively common. Dysmorphology and skeletal involvement such as those seen in ruminants are unusual. The purpose of this study is to determine the range of clinical expression in human beta-mannosidosis resulting from null mutations. We determined that the beta-mannosidase gene consists of 17 exons. Intron-based PCR primers were designed and used to amplify each of the exons in genomic DNA isolated from patient fibroblasts. We identified two patients with null mutations. Results of the analysis showed that one patient was heterozygous for nonsense mutations G334T (E83X) in exon 2 and C1363T (Q426X) in exon 10, resulting in truncation of the deduced peptide sequence from 879 to 82 and 425 amino acids, respectively. The second patient was homozygous for a deletion mutation in exon 11 (1541delAT). This deletion causes a reading frame shift and 26 out of frame amino acids before a stop codon occurs in exon 12, resulting in truncation of the deduced peptide sequence from 879 to 510 amino acids. Because disease presentation in these patients with null mutations is very variable, ranging from mild to severe, we conclude that beta-mannosidosis in humans may indeed be milder than typical of other lysosomal storage disorders.