Standardization of the hemagglutination inhibition (HAI) assay for influenza serology is challenging. Poor reproducibility of HAI results from one laboratory to another is widely cited, limiting comparisons between candidate vaccines in different clinical trials and posing challenges for licensing authorities. In this study, we standardized HAI assay materials, methods, and interpretive criteria across five geographically dispersed laboratories of a multidisciplinary influenza research network and then evaluated intralaboratory and interlaboratory variations in HAI titers by repeatedly testing standardized panels of human serum samples. Duplicate precision and reproducibility from comparisons between assays within laboratories were 99.8% (99.2% to 100%) and 98.0% (93.3% to 100%), respectively. The results for 98.9% (95% to 100%) of the samples were within 2-fold of all-laboratory consensus titers, and the results for 94.3% (85% to 100%) of the samples were within 2-fold of our reference laboratory data. Low-titer samples showed the greatest variability in comparisons between assays and between sites. Classification of seroprotection (titer > 40) was accurate in 93.6% or 89.5% of cases in comparison to the consensus or reference laboratory classification, respectively. This study showed that with carefully chosen standardization processes, high reproducibility of HAI results between laboratories is indeed achievable.T he hemagglutination inhibition (HAI) assay is the primary method for determining quantitative antibody titers for influenza virus and is widely used both for licensure of vaccines and for seroepidemiologic studies examining protection in populations (1-3). The assay relies on the ability of the hemagglutinin protein on the surface of influenza virus to bind to sialic acids on the surface of red blood cells (RBCs) (4). If the patient's serum contains antibodies that block viral attachment, this interaction is inhibited. Direct comparison of results between studies has been problematic, as the reproducibility of HAI assays between laboratories has historically been poor (5-12). These studies have shown that HAI titers reported for identical specimens in different laboratories can vary as much as 80-fold or 128-fold (9, 11), with the geometric coefficient of variation (GCV) as high as 803% (5). This variability in results may relate to differences in biological reagents, protocols, and personnel training. The use of international standards (IS) may reduce interlaboratory variability (6, 11, 13), but such reagents currently exist only for influenza A H1N1 and H5N1 clade 1 viruses (14, 15). The need for standardization of HAI assays and other laboratory methods (e.g., microneutralization [MN] assays) has been highlighted as a priority of CONSISE, the Consortium for the Standardization of Influenza Seroepidemiology (16,17). CONSISE collaborators have recently published data showing that a standardized MN protocol improves the comparability of serologic results between laboratories (18); however, the conso...

Contractile agonists may stimulate mitogenic responses in airway smooth muscle by mechanisms that involve tyrosine kinases. The role of contractile agonist-evoked activation of tyrosine kinases in contractile signaling is not clear. We addressed this issue using cultured rat airway smooth muscle cells. In these cells, serotonin (5-HT, 1 microM) caused contraction (quantitated by a decrease in cell area), which was blocked by the tyrosine kinase inhibitor genistein (40 microM). Genistein and tyrphostin 23 (40 and 10 microM, respectively) significantly decreased 5-HT-evoked peak Ca(2+) responses, and the effect of genistein could be observed in the absence of extracellular Ca(2+). The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 (30 microM) had no significant effect on peak Ca(2+) levels. Western analysis of cell extracts revealed that 5-HT caused a significant increase in tyrosine phosphorylation of proteins with molecular masses of approximately 70 kDa within 10 s of stimulation but no measurable tyrosine phosphorylation of the gamma isoform of phospholipase C (PLC-gamma). Tyrosine phosphorylation was inhibited by genistein. Furthermore, genistein (40 microM) significantly attenuated 5-HT-induced inositol phosphate production. We conclude that in airway smooth muscle contractile agonists acting on G protein-coupled receptors may activate tyrosine kinase(s), which in turn modulate calcium signaling by affecting, directly or indirectly, PLC-beta activity. It is unlikely that PLC-gamma or the mitogen-activated protein kinase pathway is involved in Ca(2+) signaling to 5-HT.

The highly inbred Fisher rat strain demonstrates both hyperresponsiveness of the airways to bronchoconstrictors and increased amounts of airway smooth muscle when compared with Lewis rats. We postulated that the excess airway smooth muscle in Fisher was attributable to a greater sensitivity to endogenous mitogenic stimuli with signaling mechanisms common to contractile stimuli. To test this possibility we investigated differences in the growth response of cultured airway smooth muscle cells (SMC) from these two strains. The Fisher SMC demonstrated an enhanced growth response to fetal bovine serum (FBS), as measured both by cell counting and [3H]thymidine incorporation. The difference in growth response was attributable to a more rapid transition in Fisher than Lewis SMC from the G0/G1 phase to the S phase of the cell cycle. When SMC growth was stimulated by either serotonin, endothelin 1, insulin, platelet-derived growth factor (PDGF), or combinations of the above factors, significant interstrain growth response differences were found only with either human PDGF (AB) or a combination of porcine PDGF (BB) and insulin. A polyclonal antibody to PDGF was less inhibitory to Fisher than Lewis SMC following stimulation with FBS. Thus, interstrain growth differences may reflect different responses to PDGF present in FBS, or to other factors which exert an influence during the G0 or G1 phase of the cell cycle.

Nitric oxide (NO) is known to be synthesized from l-arginine in a reaction catalyzed by NO synthase. Liver cytochrome P-450 enzymes also catalyze the oxidative cleavage of CN bonds of compounds containing a -C(NH2)NOH function, producing NO in vitro. The present study was designed to investigate whether there was evidence of a similar pathway for the production of NO in tracheal smooth muscle cells. Formamidoxime (10−2 to 10−4 M), a compound containing -C(NH2)NOH, relaxed carbachol-contracted tracheal rings and increased intracellular cGMP in cultured tracheal smooth muscle cells, whereasl-arginine had no such effect. NO was detectable in the medium containing cultured tracheal smooth muscle cells when incubated with formamidoxime. Ethoxyresorufin (10−7 to 10−4 M), an alternate cytochrome P-450 substrate, inhibited formamidoxime-induced cGMP accumulation as well as tracheal ring relaxation in cultured tracheal smooth muscle cells. The NO synthase inhibitors N ω-nitro-l-arginine (10−3 M) and N G-monomethyl-l-arginine (10−3 M) had no effect on formamidoxime-induced cGMP accumulation. These results suggest that NO can be synthesized from formamidoxime in tracheal smooth muscle cells, presumably by a reaction catalyzed by cytochrome P-450.