Evaluation of R-Mix shell vials for the diagnosis of viral respiratory tract infections.

Respiratory viruses cause significant morbidity and mortality. The management of these infections can be improved by a rapid diagnosis and administration of available virus-specific therapy. The goal of this study was to compare R-Mix, an engineered tissue monolayer for rapid shell vial (SV) diagnosis of viral respiratory infections, with conventional tissue culture (TC) and conventional respiratory SV (primary rhesus monkey kidney (RhMK) and Hep2 monolayers). The primary outcome measure was sensitivity for detection of influenza A and B, respiratory syncytial virus, parainfluenza 1-3, and adenovirus. The study was performed in two phases: (1) the three methods were compared using 250 nasal washes from children with lower respiratory tract infections; (2) a modified R-Mix SV harvesting schedule (SV were harvested at 24 and 120 h) was compared with TC and conventional RhMK/Hep2 SV using 311 respiratory specimens. A total of 110 viruses were identified in the first and 55 in the second phase. Diagnostic accuracies of R-Mix harvested at 24, 48, and 120 h were 98%, whereas for TC varied between 99 and 100%, and for RhMK/Hep2 SV between 98 and 99%. Sensitivities of R-Mix harvested at 24, 48, and 120 h were 26, 75, and 47%, respectively, whereas for TC varied between 60 and 94%, and for RhMK/Hep2 SV between 62 and 85%. R-Mix harvested at 48 h represent a valuable substitute for RhMK/Hep2 SV because they have comparable sensitivities and diagnostic accuracies, but R-Mix offers several technical advantages. In contrast, R-Mix harvested at 24h did not seem a very useful diagnostic tool. The utility of R-Mix harvested at 120 h, which accelerated the diagnosis of 16% of positive specimens in study phase 2, needs further investigation.     

Progenitor egress from the bone marrow after allergen challenge: role of stromal cell-derived factor 1alpha and eotaxin

BACKGROUND: CCR3 expression on CD34+ cells mediates migration to eotaxin in vitro. CXCR4 and stromal cell-derived factor (SDF)-1alpha are important for stem cell homing to hemopoietic compartments. OBJECTIVE: To study chemokine-mediated progenitor cell traffic in allergic inflammation. METHODS: Bone marrow (BM) aspirates were obtained at baseline from normal subjects; atopic subjects without asthma; and subjects with asthma before, 5 hours after, and 24 hours after allergen inhalation (dual and early responders). Changes in chemokine receptor expression and migration were assessed. RESULTS: Expression of CXCR4, but not CCR3, on BM CD34+ cells was greater in normal subjects compared with atopic subjects with asthma. Likewise, SDF-1alpha, but not eotaxin, stimulated a greater migrational response by BM CD34+ cells from normal subjects compared with subjects with asthma. For all subjects, a positive correlation was found between intensity of CXCR4 expression and magnitude of CD34+ cell response to SDF-1alpha. Allergen inhalation attenuated both intensity of CXCR4 expression and SDF-1alpha levels in marrow from dual compared with early responders 24 hours postallergen. In contrast, the intensity of CCR3 expression on BM CD34+ cells increased in dual compared with early responders at 24 hours postallergen. In addition, an increase in migrational responsiveness of BM CD34+ cells to eotaxin and a decrease to SDF-1alpha 24 hours postallergen was found in dual responder subjects with asthma. CONCLUSION: After allergen inhalation in subjects with asthma, a downregulation in CXCR4 intensity on BM CD34+ cells and a reduction in BM SDF-1alpha levels may reduce progenitor retention to marrow stroma promoting peripheral egress, possibly mediated by the CCR3/eotaxin axis.     

Adding salmeterol to an inhaled corticosteroid reduces allergen-induced serum IL-5 and peripheral blood eosinophils

BACKGROUND: Adding a long-acting beta(2)-agonist to inhaled corticosteroids results in better symptomatic asthma control than increasing the dose of inhaled corticosteroids. OBJECTIVE: Investigating whether adding the long-acting beta(2)-agonist salmeterol to the inhaled corticosteroid fluticasone propionate has an effect on allergen-induced allergic inflammation in asthma. METHODS: Bronchial allergen challenges were performed in 26 patients with allergic asthma, pretreating them with a single dose of either fluticasone/salmeterol (100/50 microg) or fluticasone alone (100 microg), in a double-blind, randomized, cross-over design. Sputum and serum markers of bronchial inflammation were measured after allergen challenge, as well as lung function parameters. Primary outcomes were sputum eosinophil numbers and eosinophil cationic protein. RESULTS: Asthmatic responses after allergen challenge were significantly reduced after pretreatment with fluticasone/salmeterol relative to fluticasone alone. Sputum inflammatory markers after allergen challenge were not significantly affected by fluticasone/salmeterol pretreatment. By contrast, serum IL-5 was significantly reduced (geometric mean serum IL-5 [SEM]: 0.5 [0.3] vs 1.1 [0.3] pg/mL 1 hour and 0.6 [0.3] vs 1.1 [0.3] pg/mL 6 hours after challenge with fluticasone/salmeterol vs fluticasone alone pretreatment, respectively; P values < .05). Also, peripheral blood eosinophils were significantly reduced (geometric mean number x 10(6)/L [SEM]: 172 [0.1] vs 237 [0.1] at 6 hours and 271 [0.1] vs 351 [0.1] at 24 hours with fluticasone/salmeterol vs fluticasone alone pretreatment, respectively; P < .05). CONCLUSION: Adding salmeterol to fluticasone reduces allergen-induced serum IL-5 and peripheral blood eosinophils. This phenomenon may contribute to the improved clinical outcomes that result from adding a long-acting beta(2)-agonist to inhaled corticosteroids.     

Clonal chromosome abnormalities in human breast carcinomas I. Twenty-eight cases with primary disease

Cytogenetic analysis was performed on a selected series of short-term cultures of primary breast carcinomas from 28 patients. All patients had histopathologically confirmed malignancies, with the majority (25/28 cases) demonstrating infiltrating ductal carcinoma. All 28 cases evidenced clonal chromosome abnormalities, with 10/28 displaying only numeric aberrations, whereas 18/28 displayed clonal structural alterations. In near-diploid tumors the most common numeric changes were — 17 and — 19. However, trisomy 7 was the only numeric change in two near-diploid tumors. Structural chromosome alterations were primarily isochromosomes, apparent terminal deletions, and unbalanced non-reciprocal translocations. Chromosomes 1 (10/18–56%) and 6 (8/18–44%) were most frequently altered in this series. Breakpoints of clonal structural abnormalities were shown to cluster to several chromosome segments, including 1p22-q11, 3p11, 6p11–13, 7p11-q11, 8p11-q11, and 19q13. Analysis of the gain or loss of specific chromosome segments revealed that the most consistent tendency was over-representation of 1q, 3q, and 6p. © 1993 Wiley-Liss, Inc.     

Effect of marrow on the high frequency ultrasonic properties of cancellous bone

A number of investigators have performed in vitro measurements of cancellous bone to determine how various ultrasonic parameters depend on bone density and trabecular orientation. To facilitate handling and storage of bone specimens, the marrow is often removed prior to ultrasonic measurements. However, the assumption that marrow does not affect ultrasonic measurements at high frequencies (>1 MHz) has not been tested. The goal of this study is to determine the effect of marrow on the ultrasonic properties of bovine cancellous bone at frequencies greater than 1 MHz. Twelve specimens of cancellous bone were obtained from the proximal end of four bovine tibia. Ultrasonic measurements consisting of normalized broadband ultrasonic attenuation (nBUA), speed of sound (SOS) and apparent integrated backscatter (AIB) were measured in each specimen using 2.25 MHz (centre frequency) broadband ultrasonic pulses. These measurements were performed before and after marrow removal either along the superoinferior (SI) or mediolateral (ML) direction. SOS and nBUA showed no significant difference (p > 0.05) for either direction of propagation after marrow removal. AIB showed no significant difference in the SI direction. For the ML direction, a small but statistically significant difference (p = 0.044) was observed after marrow removal.     

Prevention of polyglutamine oligomerization and neurodegeneration by the peptide inhibitor QBP1 in Drosophila

Polyglutamine (polyQ) diseases are a growing class of inherited neurodegenerative diseases including Huntington's disease, which are caused by abnormal expansions of the polyQ stretch in each unrelated disease protein. The expanded polyQ stretch is thought to confer toxic properties on the disease proteins through alteration of their conformation leading to pathogenic protein-protein interactions including oligomerization and/or aggregation. Hypothesizing that molecules with selective binding affinity to the expanded polyQ stretch may interfere with the pathogenic properties, we previously identified Polyglutamine Binding Peptide 1 (QBP1) from combinatorial peptide phage display libraries. We show here that a tandem repeat of the inhibitor peptide QBP1, (QBP1)(2), significantly suppresses polyQ aggregation and polyQ-induced neurodegeneration in the compound eye of Drosophila polyQ disease models, which express the expanded polyQ protein under the eye specific promoter. Most importantly, (QBP1)(2) expression dramatically rescues premature death of flies expressing the expanded polyQ protein in the nervous system, resulting in the dramatic increase of the median life span from 5.5 to 52 days. These results suggest that QBP1 can prevent polyQ-induced neurodegeneration in vivo. We propose that QBP1 prevents polyQ oligomerization and/or aggregation either by altering the toxic conformation of the expanded polyQ stretch, or by simply competing with the expanded polyQ stretches for binding to other expanded polyQ proteins. The peptide inhibitor QBP1 is a promising candidate with great potential as a therapeutic molecule against the currently untreatable polyQ diseases.     

Receptor-binding properties of modern human influenza viruses primarily isolated in Vero and MDCK cells and chicken embryonated eggs

To study the receptor specificity of modern human influenza H1N1 and H3N2 viruses, the analogs of natural receptors, namely sialyloligosaccharides conjugated with high molecular weight (about 1500 kDa) polyacrylamide as biotinylated and label-free probes, have been used. Viruses isolated from clinical specimens were grown in African green monkey kidney (Vero) or Madin-Darby canine kidney (MDCK) cells and chicken embryonated eggs. All Vero-derived viruses had hemagglutinin (HA) sequences indistinguishable from original viruses present in clinical samples, but HAs of three of seven tested MDCK-derived isolates had one or two amino acid substitutions. Despite these host-dependent mutations and differences in the structure of HA molecules of individual strains, all studied Vero- and MDCK-isolated viruses bound to Neu5Ac alpha2-6Galbeta1-4GlcNAc (6'SLN) essentially stronger than to Neu5Acalpha2-6Galbeta1-4Glc (6'SL). Such receptor-binding specificity has been typical for earlier isolated H1N1 human influenza viruses, but there is a new property of H3N2 viruses that has been circulating in the human population during recent years. Propagation of human viruses in chicken embryonated eggs resulted in a selection of variants with amino acid substitutions near the HA receptor-binding site, namely Gln226Arg or Asp225Gly for H1N1 viruses and Leu194Ile and Arg220Ser for H3N2 viruses. These HA mutations disturb the observed strict 6'SLN specificity of recent human influenza viruses.     

Induction of interleukin 2 (IL2) and interferon-γ and enhancement of IL2 receptor expression by a CD26 monoclonal antibody

The ability of the 134-2C2 monoclonal antibody (mAb; CD26) to transmit an activation signal and to affect T cell proliferation has been studied. The 134-2C2 mAb, although not being mitogenic by itself, is able to increase the proliferation of purified T cells in the presence of exogenous interleukin 2 (IL2) or phorbol 12-myristate 13-acetate (PMA). No effect of our mAb was observed on the proliferation of T cells induced by other stimuli such as Sepharose-bound CD3 mAb, phytohemagglutinin or calcium ionophore. Since the co-stimulatory effect of 134-2C2 mAb on PMA-induced T cell proliferation was strongly inhibited by an anti-Tac antibody, its involvement on the IL2/IL2 receptor pathway was investigated. An increased IL2 secretion in T cells cultured with PMA plus 134-2C2 mAb was observed and Northern blot analysis showed that the mAb 134-2C2 acts synergistically with PMA favoring the induction of both IL2 and interferon-γ mRNA synthesis, as well as the enhancement of IL2 receptor and transferrin receptor mRNA expression. Studies on mechanisms implicated in signal transduction showed that 134-2C2 mAb modifies neither intracellular calcium levels nor phosphoinositide breakdown. Additionally, no effect was exerted on protein kinase C translocation. These data suggest that the CD26 antigen is involved in T cell activation in an IL2/IL2 receptor-dependent pathway.     

Domains of macaque DC-SIGN essential for capture and transfer of simian immunodeficiency virus

The C-type lectin DC-SIGN mediates the capture and transfer of simian immunodeficiency virus (SIV) from macaque dendritic cells (DCs) to permissive T-cells. To further identify the determinants in macaque DC-SIGN required for capture and transfer of virus, we created mutants containing deletions or point mutations in the extracellular domains, and tested their ability to capture and transmit SIV. We found that SIV bound to the carbohydrate recognition domain (CRD) of macaque DC-SIGN via the envelope protein. In addition, deleting the C-terminal half of the CRD, or mutating amino acids within this region that contact Ca(2+) or mannose, disrupted virion capture activity. However, an N-terminal CRD deletion mutant was capable of binding SIV, indicating that this region was not necessary for binding. Finally, deletion of the neck domain also reduced the capacity for macaque DC-SIGN to capture SIV. Interestingly, ICAM-3, the cellular ligand for DC-SIGN, did not bind to any of the DC-SIGN mutants, including mutants with amino acid changes in the N-terminal region of the CRD. These data suggest that the binding sites for SIV and ICAM-3 may be distinct but overlapping. Together, the data demonstrate the importance of both the neck and the CRD of macaque DC-SIGN for efficient capture of SIV and binding to ICAM-3.