A rapid influenza diagnostic test based on detection of viral neuraminidase activity.

Current methods used for diagnosis of acute infection of pathogens rely on detection of nucleic acids, antigens, or certain classes of antibodies such as IgM. Here we report a virus enzyme assay as an alternative to these methods for detection of acute viral infection. In this method, we used a luciferin derivative as the substrate for detection of the enzyme activity of influenza viral neuraminidase as a means for diagnosis of influenza. The resulting commercial test, the qFLU Dx Test, uses a different supply chain that does not compete with those for the current tests. The assay reagents were formulated as a master mix that accommodated both the neuraminidase and luciferase reactions, thereby enabling rapid and prolonged production of stable light signal in the presence of influenza virus in the sample. The assay was evaluated using depository throat swab specimens. As expected, the assay exhibited similar detection rates for all influenza types and subtypes except for A(H7N9), which exhibited lower detection rate due to lower viral titer in the specimens. When throat swab specimens were diluted with the sample buffer of the test kit and tested with the qFLU Dx Test. The sensitivity and specificity were 82.41% (95% confidence interval: 79.66-85.84%) and 95.39% (95% confidence interval: 94.32-96.46%), respectively, for these diluted specimens in comparison to a real-time polymerase chain reaction assay. The uniqueness of the qFLU Dx Test as an enzymatic assay makes it highly complementary with currently available methods.

A Biochemiluminescent Sialidase Assay for Diagnosis of Bacterial Vaginosis.

Bacterial vaginosis (BV) is a common condition among women of reproductive age. A sensitive, quantitative and rapid assay is needed for the diagnosis of and, particularly, therapy monitoring for BV. Bacterial sialidase appears to play an important role in bacterial biofilms on vaginal epithelium, a condition closely associated with BV. Here, we report a biochemiluminescent sialidase assay that uses a substrate derivatized with firefly luciferin. In the presence of sialidase in the reaction, the substrate is cleaved to release luciferin, which is subsequently oxidized by firefly luciferase to generate a light signal. Thus, the light signal intensity can be used to detect and measure the relative concentration of sialidase in a vaginal sample as a means of BV diagnosis. All reagents are present in a reagent bead and sample buffer, enabling essentially a one-step assay. The assay is highly sensitive and quantitative, with a sensitivity and specificity of 95.40% and 94.94%, respectively, compared to the Amsel method. Interestingly, only 27.6% of those with BV had high levels of sialidase activity with a signal to cutoff ratio of 10 or more. The assay may be used for diagnosis of BV, risk assessment of BV patients in terms of sialidase activity levels, and monitoring antibiotic therapy.

Targeting minor histocompatibility antigens in graft versus tumor or graft versus leukemia responses.

Allogeneic hematopoietic cell transplantation (alloHCT) represents the only curative therapy for several hematologic malignancies, and shows promise as a nascent treatment modality for select solid tumors. Although the original goal of alloHCT was hematopoietic reconstitution after sub-lethal chemoradiotherapy, recognition of a profound donor lymphocyte-mediated graft-versus-leukemia (GVL) or graft-versus-tumor (GVT) effect has shifted the paradigm from pre-transplant cytoreduction to tumor control via donor lymphocytes. In human leukocyte antigen (HLA)-compatible alloHCT, GVL and GVT reactions are induced primarily by donor T-cell recognition of minor histocompatibility antigens (mHAgs). Here we review the literature regarding mHAg-specific T cells in GVL and GVT reactions, and discuss the prospects of exploiting mHAgs as immunotherapeutic targets.

ELMO1 and Dock180, a bipartite Rac1 guanine nucleotide exchange factor, promote human glioma cell invasion.

A distinct feature of malignant gliomas is the intrinsic ability of single tumor cells to disperse throughout the brain, contributing to the failure of existing therapies to alter the progression and recurrence of these deadly brain tumors. Regrettably, the mechanisms underlying the inherent invasiveness of glioma cells are poorly understood. Here, we report for the first time that engulfment and cell motility 1 (ELMO1) and dedicator of cytokinesis 1 (Dock180), a bipartite Rac1 guanine nucleotide exchange factor (GEF), are evidently linked to the invasive phenotype of glioma cells. Immunohistochemical analysis of primary human glioma specimens showed high expression levels of ELMO1 and Dock180 in actively invading tumor cells in the invasive areas, but not in the central regions of these tumors. Elevated expression of ELMO1 and Dock180 was also found in various human glioma cell lines compared with normal human astrocytes. Inhibition of endogenous ELMO1 and Dock180 expression significantly impeded glioma cell invasion in vitro and in brain tissue slices with a concomitant reduction in Rac1 activation. Conversely, exogenous expression of ELMO1 and Dock180 in glioma cells with low level endogenous expression increased their migratory and invasive capacity in vitro and in brain tissue. These data suggest that the bipartite GEF, ELMO1 and Dock180, play an important role in promoting cancer cell invasion and could be potential therapeutic targets for the treatment of diffuse malignant gliomas.

CRIT peptide interacts with factor B and interferes with alternative pathway activation.

Complement C2 receptor inhibitor trispanning (CRIT) inhibits the classical pathway (CP) C3 convertase formation by competing with C4b for the binding of C2. The C-terminal 11-amino-acid of the first CRIT-extracellular domain (CRIT-H17) has a strong homology with a sequence in the C4beta chain, which is responsible for the binding of C2. Since the CP and alternative pathway (AP) C3 convertases have many functional and structural similarities, we further investigated the effects of CRIT-H17 on the AP. The factor D-mediated cleavage of factor B (FB) was blocked by CRIT-H17. By ELISA and immunoblot, CRIT-H17 was shown to bind FB. CRIT-H17 had no decay activity on the C3bBb complex as compared to decay-accelerating factor. Binding of CRIT-H17 to FB did not interfere with the assembly of C3bB complex. In a haemolytic assay using C2-deficient serum, CRIT-H17 interfered with AP complement activation.

Expression of functional recombinant von Willebrand factor-A domain from human complement C2: a potential binding site for C4 and CRIT.

CRIT (complement C2 receptor inhibitor trispanning) is a newly described transmembrane molecule that is capable of binding C2 via its first extracellular domain (ed1). CRIT competes with C4b for the binding of C2. Previous experiments have suggested that a major binding site for C2 is located on short, almost identical peptide sequences of CRIT-ed1 and the beta-chain of C4. The C2 domains involved in binding, however, remain unknown. We cloned the vWFA (von Willebrand factor-A) domain of C2, as it is a region likely to be involved in interactions with other proteins, and were able to functionally express the 25 kDa human complement C2 vWFA domain (amino acids 224-437). The recombinant vWFA protein fixed on MagneHis Ni-Particles bound C4 in normal human serum. The C4 alpha, beta and gamma chains were separated by SDS/PAGE and purified separately by electro-elution. The purified C4 chains were then used in a sandwich ELISA, which showed the vWFA to bind C4 only via the C4beta chain. In a haemolytic assay, the recombinant vWFA protein inhibited complement activation by the classical pathway in a dose-dependent manner by competing with native C2 for binding to C4b. vWFA bound the ed1 peptide of CRIT as well, and specifically to the 11-amino-acid peptide fragment of ed1 that is known to interact with whole C2. These findings show that the vWFA domain is centrally involved in the C2-CRIT and C2-C4b bindings. The cloned vWFA domain will allow us to dissect out the fine interactions between C2 and CRIT or C4b.

Complement C2 receptor inhibitor trispanning: a novel human complement inhibitory receptor.

The complement system presents a powerful defense against infection and is tightly regulated to prevent damage to self by functionally equivalent soluble and membrane regulators. We describe complement C2 receptor inhibitor trispanning (CRIT), a novel human complement regulatory receptor, expressed on hemopoietic cells and a wide range of tissues throughout the body. CRIT is present in human parasites through horizontal transmission. Serum complement component C2 binds to the N-terminal extracellular domain 1 of CRIT, which, in peptide form, blocks C3 convertase formation and complement-mediated inflammation. Unlike C1 inhibitor, which inhibits the cleavage of C4 and C2, CRIT only blocks C2 cleavage but, in so doing, shares with C1 inhibitor the same functional effect, of preventing classical pathway C3 convertase formation. Ab blockage of cellular CRIT reduces inhibition of cytolysis, indicating that CRIT is a novel complement regulator protecting autologous cells.

Naturally occurring 2'-hydroxyl-substituted flavonoids as high-affinity benzodiazepine site ligands.

Screening of traditional medicines has proven invaluable to drug development and discovery. Utilizing activity-guided purification, we previously reported the isolation of a list of flavonoids from the medicinal herb Scutellaria baicalensis Georgi, one of which manifested an affinity for the benzodiazepine receptor (BDZR) comparable to that of the synthetic anxiolytic diazepam (K(i)=6.4 nM). In the present study, this high-affinity, naturally occurring flavonoid derivative, 5,7,2'-trihydroxy-6,8-dimethoxyflavone (K36), was chosen for further functional and behavioral characterization. K36 inhibited [3H]flunitrazepam binding to native BDZR with a K(i) value of 6.05 nM. In electrophysiological experiments K36 potentiated currents mediated by rat recombinant alpha(1)beta(2)gamma(2) GABA(A) receptors expressed in Xenopus oocytes. This potentiation was characterized by a threshold (1 nM) and half-maximal stimulation (24 nM) similar to diazepam. This enhancement was demonstrated to act via the BDZR, since co-application of 1 microM of the BDZR antagonist Ro15-1788 reversed the potentiation. Oral administration of K36 produced significant BDZR-mediated anxiolysis in the mice elevated plus-maze, which was abolished upon co-administration of Ro15-1788. Sedation, myorelaxation and motor incoordination were not observed in the chosen dosage regimen. Structure-activity relationships utilizing synthetic flavonoids with different 2' substituents on the flavone backbone supported that 2'-hydroxyl-substitution is a critical moiety on flavonoids with regard to BDZR affinities. These results further underlined the potential of flavonoids as therapeutics for the treatment of BDZR-associated syndromes.

Structure-activity relationships of flavonoids, isolated from Scutellaria baicalensis, binding to benzodiazepine site of GABA(A) receptor complex.

Twenty-six flavonoids were isolated from Scutellaria baicalensis. Their affinities for the benzodiazepine (BDZ) binding site of GABA A receptor have been studied using [ 3H]flunitrazepam binding to rat cortical membranes in vitro. The structure-activity relationships suggested that 2'-OH flavones exhibited the most potent binding affinity, which could lead to the design and discovery of new BDZ receptor ligands.

Anxiolytic effect of wogonin, a benzodiazepine receptor ligand isolated from Scutellaria baicalensis Georgi.

The search for novel anxiolytics devoid of undesirable side-effects typical of classical benzodiazepines (BDZs) has been intense, and flavonoids, as a relative new class of ligands, have been shown to possess anxiolytic effects in vivo. The present study evaluated the pharmacological properties of a naturally occurring monoflavonoid, 5,7-dihydroxy-8-methoxyflavone or wogonin. The affinity (K(i)) of wogonin for the benzodiazepine site (BZD-S) on the gamma-aminobutyric acid(A) (GABA(A)) receptor complex was 0.92 microM. Using electrophysiological techniques, we showed that wogonin enhanced the GABA-activated current in rat dorsal root ganglion neurons, and in Xenopus laevis oocytes expressing recombinant rat GABA(A) receptors, the enhancement was partially reversed by the co-application of a 1 microM concentration of the BZD-S antagonist anexate (Ro15-1788). Acute toxicity and behavioral effects were examined in mice. Acute lethal activity was low, with an LD(50) of 3.9 g/kg. Oral administration of wogonin (7.5 to 30 mg/kg) elicited an anxiolytic response that was similar to that elicited by diazepam in the elevated plus-maze; a dose-dependent increase in open arm entries and time spent in open arms was observed. More importantly, its anxiolytic effect was blocked by the co-administration of Ro15-1788. In the holeboard test, not only did wogonin-treated mice experience an increased number of head-dips but they also spent more time at it, showing no signs of sedation. Furthermore, wogonin did not cause myorelaxant effects in the horizontal wire test. Taken together, these data suggest that wogonin exerts its anxiolytic effect through positive allosteric modulation of the GABA(A) receptor complex via interaction at the BZD-S. Its anxiolytic effect was not accompanied by sedative and myorelaxant side-effects typical of BDZs.

Two flavones from Scutellaria baicalensis Georgi and their binding affinities to the benzodiazepine site of the GABAA receptor complex.

A new flavone 6,2'-dihydroxy-5,7,8,6'-tetramethoxyflavone (1) together with one known flavone 5,7,2'-trihydroxy-6,8-dimethoxyflavone (2) were isolated from the roots of Scutellaria baicalensis Georgi. Their structures were elucidated on the basis of spectral evidence and their affinities for the benzodiazepine (BDZ) site of the GABAA receptor complex were evaluated with a radioligand receptor binding assay.