Fabrication of an electrochemical sensor based on carbon nanotubes modified with gold nanoparticles for determination of valrubicin as a chemotherapy drug: valrubicin-DNA interaction.

In this study, an electrochemical sensor was fabricated based on gold nanoparticles/ ethylenediamine/ multi-wall carbon-nanotubes modified gold electrode (AuNPs/en/MWCNTs/AuE) for determination of valrubicin in biological samples. Valrubicin was effectively accumulated on the surface of AuNPs/en/MWCNTs/AuE and produced a pair of redox peaks at around 0.662 and 0.578V (vs. Ag/AgCl) in citrate buffer (pH4.0). The electrochemical parameters including pH, buffer, ionic strength, scan rate and size of AuNPs have been optimized. There was a good linear correlation between cathodic peak current and concentration of valrubicin in the range of 0.5 to 80.0µmolL(-1) with the detection limit of 0.018µmolL(-1) in citrate buffer (pH4.0) and 0.1molL(-1) KCl. Finally, the constructed sensor was successfully applied for determination of valrubicin in human urine and blood serum. In further studies, the different sequences of single stranded DNA probes have been immobilized on the surface of AuNPs decorated on MWCNTs to study the interaction of oligonucleotides with valrubicin.

Management of paediatric tuberculosis in provincial and district hospitals in Afghanistan.

Case detection, diagnosis and treatment of tuberculosis 1 B) in children are challenging issues vorldwide. This study in Afghanistan aimed to evaluate paediatric TB case management, including contact investigation, at health facilities where all diagnostic processes were available. In 7 out of 8 regions of the country 1 province was selected. Documents used for management of paediatric TB cases were reviewed in 15 distinct hospitals and 8 provincial hospitals in the selected provinces. The key issues which emerged were: a low suspect rate among total outpatients (0.4%) and a very low suspect rate among children aged < 5 years; low performance of suspect management (68.5% suspects received further examinations); low utilization of other diagnostic methods; a high early defaulter rate (14.0%); and insufficient coverage of contact management (74.0%). This survey indicated that the Afghanistan national TB programme needs to develop plans to improve the quality of diagnosis, suspect management and contact management in paediatric TB cases.

Management of paediatric tuberculosis in provincial and district hospitals in Afghanistan

Case detection, diagnosis and treatment of tuberculosis [TB] in children are challenging issues worldwide. This study in Afghanistan aimed to evaluate paediatric TB case management, including contact investigation, at health facilities where all diagnostic processes were available. In 7 out of 8 regions of the country 1 province was selected. Documents used for management of paediatric TB cases were reviewed in 15 distinct hospitals and 8 provincial hospitals in the selected provinces. The key issues which emerged were: a low suspect rate among total outpatients [0.4%] and a very low suspect rate among children aged < 5 years; low performance of suspect management [68.5% suspects received further examinations]; low utilization of other diagnostic methods; a high early defaulter rate [14.0%]; and insufficient coverage of contact management [74.0%]. This survey indicated that the Afghanistan national TB programme needs to develop plans to improve the quality of diagnosis, suspect management and contact management in paediatric TB cases

Mapping of IFN-beta epitopes important for receptor binding and biologic activation: comparison of results achieved using antibody-based methods and alanine substitution mutagenesis.

The epitopes important for receptor binding and activation of human interferon-beta1a (IFN-beta1a) were mapped with monoclonal antibodies (mAb), grouped on the basis of their specificity and ability to neutralize biologic activity, and alanine scanning mutagenesis (ASM). The binding properties of nine mAb were defined, using ASM-IFN-beta mutants having alanine substituted at targeted, surface-exposed residues. The results were correlated with the mAb neutralizing potency. Of six mAb that bound either at or adjacent to the IFNAR-2 receptor chain binding site defined by the ASM epitopes, only three had measurable neutralizing activity. Two of these inhibited IFN-beta/IFNAR-2 complex formation, suggesting that steric hindrance of receptor binding constitutes their mechanism of neutralization. However, two mAb that bound to sites remote from the IFNAR-2 binding site on IFN-beta also inhibited IFN-beta/IFNAR-2 complex formation and demonstrated potent neutralizing activity. Thus, neutralizing mAb may employ mechanisms other than steric blockade to inhibit directly the binding of receptor by cytokine, limiting their usefulness as tools to define precise receptor-ligand interaction sites.

BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF.

B cell homeostasis has been shown to critically depend on BAFF, the B cell activation factor from the tumor necrosis factor (TNF) family. Although BAFF is already known to bind two receptors, BCMA and TACI, we have identified a third receptor for BAFF that we have termed BAFF-R. BAFF-R binding appears to be highly specific for BAFF, suggesting a unique role for this ligand-receptor interaction. Consistent with this, the BAFF-R locus is disrupted in A/WySnJ mice, which display a B cell phenotype qualitatively similar to that of the BAFF-deficient mice. Thus, BAFF-R appears to be the principal receptor for BAFF-mediated mature B cell survival.

BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population.

The tumor necrosis factor (TNF) family member B cell activating factor (BAFF) binds B cells and enhances B cell receptor-triggered proliferation. We find that B cell maturation antigen (BCMA), a predicted member of the TNF receptor family expressed primarily in mature B cells, is a receptor for BAFF. Although BCMA was previously localized to the Golgi apparatus, BCMA was found to be expressed on the surface of transfected cells and tonsillar B cells. A soluble form of BCMA, which inhibited the binding of BAFF to a B cell line, induced a dramatic decrease in the number of peripheral B cells when administered in vivo. Moreover, culturing splenic cells in the presence of BAFF increased survival of a percentage of the B cells. These results are consistent with a role for BAFF in maintaining homeostasis of the B cell population.

Systematic mutational mapping of sites on human interferon-beta-1a that are important for receptor binding and functional activity.

A systematic mutational analysis of human interferon-beta-1a (IFN-beta) was performed to identify regions on the surface of the molecule that are important for receptor binding and for functional activity. The crystal structure of IFN-beta-1a was used to design a panel of 15 mutant proteins, in each of which a contiguous group of 2-8 surface residues was mutated, in most instances to alanine. The mutants were analyzed for activity in vitro in antiviral and in antiproliferation assays, and for their ability to bind to the type I IFN (ifnar1/ifnar2) receptor on Daudi cells and to a soluble ifnar2 fusion protein (ifnar2-Fc). Abolition of binding to ifnar2-Fc for mutants A2, AB1, AB2, and E established that the ifnar2 binding site on IFN-beta comprises parts of the A helix, the AB loop, and the E helix. Mutations in these areas, which together define a contiguous patch of the IFN-beta surface, also resulted in reduced affinity for binding to the receptor on cells and in reductions in activity of 5-50-fold in functional assays. A second receptor interaction site, concluded to be the ifnar1 binding site, was identified on the opposite face of the molecule. Mutations in this region, which encompasses parts of the B, C, and D helices and the DE loop, resulted in disparate effects on receptor binding and on functional activity. Analysis of antiproliferation activity as a function of the level of receptor occupancy allowed mutational effects on receptor activation to be distinguished from effects on receptor binding. The results suggest that the binding energy from interaction of IFN-beta with ifnar2 serves mainly to stabilize the bound IFN/receptor complex, whereas the binding energy generated by interaction of certain regions of IFN-beta with ifnar1 is not fully expressed in the observed affinity of binding but instead serves to selectively stabilize activated states of the receptor.

Divalent cations stabilize the alpha 1 beta 1 integrin I domain.

Recent structural and functional analyses of alpha integrin subunit I domains implicate a region in cation and ligand binding referred to as the metal ion-dependent adhesion site (MIDAS). Although the molecular interactions between Mn2+ and Mg2+ and the MIDAS region have been defined by crystallographic analyses, the role of cation in I domain function is not well understood. Recombinant alpha 1 beta 1 integrin I domain (alpha1-I domain) binds collagen in a cation-dependent manner. We have generated and characterized a panel of antibodies directed against the alpha1-I domain, and selected one (AJH10) that blocks alpha 1 beta 1 integrin function for further study. The epitope of AJH10 was localized within the loop between the alpha 3 and alpha 4 helices which contributes one of the metal coordination sites of the MIDAS structure. Kinetic analyses of antibody binding to the I domain demonstrate that divalent cation is required to stabilize the epitope. Denaturation experiments demonstrate that cation has a dramatic effect on the stabilization of the I domain structure. Mn2+ shifts the point at which the I domain denatures from 3.4 to 6.3 M urea in the presence of the denaturant, and from 49.5 to 58.6 degrees C following thermal denaturation. The structural stability provided to the alpha1-I domain by divalent cations may contribute to augmented ligand binding that occurs in the presence of these cations.

Characterization of antihuman IFNAR-1 monoclonal antibodies: epitope localization and functional analysis.

The type I interferon receptor (IFNAR) is composed of two subunits, IFNAR-1 and IFNAR-2, encoding transmembrane polypeptides. IFNAR-2 has a dominant role in ligand binding, but IFNAR-1 contributes to binding affinity and to differential ligand recognition. A panel of five monoclonal antibodies (mAb) to human IFNAR-1 (HuIFNAR-1) was produced and characterized. The reactivity of each mAb toward HuIFNAR-1 on native and transfected cells and in Western blot and ELISA formats was determined. In functional assays, one mAb, EA12, blocked IFN-a2 binding to human cells and interfered with Stat activation and antiviral activity. Epitopes for the mAb were localized to subdomains of the HuIFNAR-1 extracellular domain by differential reactivity of the mAb to a series of human/bovine IFNAR-1 chimeras. The antibody EA12 seems to require native HuIFNAR-1 for reactivity and does not map to a single subdomain, perhaps recognizing an epitope containing noncontiguous sequences in at least two subdomains. In contrast, the epitopes of the non-neutralizing mAb FB2, AA3, and GB8 mapped, respectively, to the first, second, and third subdomains of HuIFNAR-1. The mAb DB2 primarily maps to the fourth subdomain, although its reactivity may be affected by other determinants.

The murine anti-human common gamma chain monoclonal antibody CP.B8 blocks the second step in the formation of the intermediate affinity IL-2 receptor.

A murine monoclonal antibody, CP.B8, specific for the extracellular portion of the human common gamma (gammac) chain, and its Fab fragment are shown to block the binding of IL-2 to COS-7 cells transfected with the cDNA for the full-length IL-2 receptor beta (IL-2Rbeta) and gammac chains, components which together comprise the intermediate affinity IL-2 receptor (IL-2R) expressed on the surface of resting T cells, NK cells, and on certain intestinal epithelial cells. To investigate the mechanism of this inhibition, the extracellular portions of the IL-2Rbeta and gammac chains were expressed and purified, and their interactions with each other and with IL-2 were studied by gel filtration and by surface plasmon resonance (SPR). By gel filtration, a stable ternary complex was formed by association of the three proteins, while no stable binary complexes were detected between any two of the three proteins. By SPR analysis, IL-2 was shown to associate rapidly with IL-2Rbeta, forming a binary complex with an equilibrium dissociation constant (Kd) of 800 nM, which permitted subsequent association of the gammac chain. Dissociation of the IL-2/IL-2Rbeta/gammac chain complex was significantly slower than dissociation of the IL-2/IL-2Rbeta complex. Using these model systems, we tested the ability of mAb CP.B8 to inhibit the association of the gammac chain with IL-2 and IL-2Rbeta. By gel filtration, mAb CP.B8 formed a stable complex with the gammac chain, preventing its association with IL-2 and IL-2Rbeta. MAb CP.B8 was also capable of dissociating the gammac chain already complexed with IL-2 and IL-2Rbeta. SPR analysis confirmed these findings and showed, in addition, that the Fab fragment of CP.B8 was also capable of inhibiting the association of the gammac chain with the IL-2/IL-2Rbeta complex. We conclude that mAb CP.B8 blocks the second step in the formation of the intermediate affinity IL-2R on the surface of transfected COS-7 cells by binding at or close to a region on the gammac chain that is involved in contact with IL-2 and/or IL-2Rbeta.

Cytotoxic activities of recombinant soluble murine lymphotoxin-alpha and lymphotoxin-alpha beta complexes.

Human lymphotoxin-alpha (LT alpha) is found in a secreted form and on the surface of lymphocytes as a complex with a second related protein called lymphotoxin-beta (LT beta). Both secreted human LT alpha and TNF have similar biological activities mediated via the TNF receptors, whereas the cell surface LT alpha beta complex binds to a separate receptor called the LT beta receptor (LT beta R). The murine LT alpha and LT beta (mLT alpha and mLT beta) proteins have never been characterized. When recombinant mLT alpha was produced by either of several methods, the protein had a very low specific activity relative to that of human LT alpha in the conventional WEHI 164 cytotoxicity bioassay. The weak activity observed was inhibited by a soluble murine TNF-R55 Ig fusion protein (mTNF-R55-Ig), but not by mLT beta R-Ig. Coexpression of both mLT alpha and a soluble version of mLT beta in insect cells led to an LT alpha beta form that was cytotoxic in the WEHI 164 assay via the LT beta R. To determine whether natural mLT alpha-like forms with cytotoxic activity comparable to that of secreted human LT alpha were secreted from primary spleen cells, splenic lymphocytes were activated in various ways, and their supernatants were analyzed for cytotoxic activity. Using specific Abs to distinguish between mTNF and mLT, a TNF component was readily detected; however, there was no evidence for a secreted mLT alpha cytotoxic activity using this assay. Combined, these observations suggest that secreted mLT alpha may not play a role in the mouse via interactions with TNF-R55, and the ramifications of this hypothesis are discussed.

Signaling through the lymphotoxin beta receptor induces the death of some adenocarcinoma tumor lines.

Surface lymphotoxin (LT) is a heteromeric complex of LT-alpha and LT-beta chains that binds to the LT-beta receptor (LT-beta-R), a member of the tumor necrosis factor (TNF) family of receptors. The biological function of this receptor-ligand system is poorly characterized. Since signaling through other members of this receptor family can induce cell death, e.g., the TNF and Fas receptors, it is important to determine if similar signaling events can be communicated via the LT-beta-R. A soluble form of the surface complex was produced by coexpression of LT-alpha and a converted form of LT-beta wherein the normally type II LT-beta membrane protein was changed to a type I secreted form. Recombinant LT-alpha 1/beta 2 was cytotoxic to the human adenocarcinoma cell lines HT-29, WiDr, MDA-MB-468, and HT-3 when added with the synergizing agent interferon (IFN) gamma. When immobilized on a plastic surface, anti-LT-beta-R monoclonal antibodies (mAbs) induced the death of these cells, demonstrating direct signaling via the LT-beta-R. Anti-LT-beta-R mAbs were also identified that inhibited ligand-induced cell death, whereas others were found to potentiate the activity of the ligand when added in solution. The human WiDr adenocarcinoma line forms solid tumors in immunocompromised mice, and treatment with an anti-LT-beta-R antibody combined with human IFN-gamma arrested tumor growth. The delineation of a biological signaling event mediated by the LT-beta-R opens a window for further studies on its immunological role, and furthermore, activation of the LT-beta-R may have an application in tumor therapy.

Characterization of surface lymphotoxin forms. Use of specific monoclonal antibodies and soluble receptors.

Lymphotoxin (LT) is a cytokine related to TNF, found in human systems in both secreted and membrane bound forms. The well characterized secreted form is a trimer of a single protein, LT-alpha, whereas the surface form is composed of a complex between two related molecules, LT-alpha and LT-beta. Because there is a distinct receptor for the complex, the membrane form is believed to signal via events different from those elicited by TNF and secreted LT-alpha. By using a battery of anti-LT-alpha and LT-beta mAbs, it is clear that two LT surface forms exist on the surface of PMA-activated II-23 cells, a human T cell hybridoma. Assuming that these surface forms are trimers, a minor form appears early after induction having an apparent stoichiometry of LT-alpha 2/beta 1 and is recognized by one group of anti-LT-alpha mAbs and the p55-TNF receptor. The second and predominant form has an apparent LT-alpha 1/beta 2 composition and is recognized by a second group of pantrophic anti-LT-alpha mAbs and the LT-beta receptor. Neither of the heteromeric forms nor a putative LT-beta homotrimeric form were found to be secreted. The properties of surface LT on the II-23 cell system were similar to those of the surface LT forms on Chinese hamster ovary cells transfected with both LT-alpha and LT-beta genes and a number of lymphoid tumor lines. These experiments point toward the LT-alpha 1/beta 2 complex as the predominant membrane form of LT on the lymphocyte surface, and this complex is the primary ligand for the LT-beta receptor.

Human type I interferon receptor, IFNAR, is a heavily glycosylated 120-130 kD membrane protein.

Type I interferons (IFNs) bind and signal through cell surface receptors that share at least one common component. One candidate for such a component is the interferon-alpha receptor (IFNAR). Genetic studies have shown that the IFNAR gene product is required for response to many type I interferons. However, these studies also suggest that the IFNAR protein interacts with an additional receptor component(s) to form functionally complete type I IFN receptors. Although these genetic studies have contributed significantly to understanding the type I IFN receptors. Although these genetic studies have contributed significantly to understanding the type I IFN receptors, little biochemical characterization of IFNAR and its function has been reported. To facilitate biochemical studies of the IFNAR gene product, a monoclonal antibody, GB8, recognizing the extracellular domain of IFNAR was prepared. The epitope for GB8 maps to the second extracellular domain of IFNAR between amino acids 278 and 293. GB8 identifies IFNAR in western blots of cell membranes as a broad band with molecular mass ranging from 100 to 150 kD in membranes from CHO cells overexpressing the human IFNAR gene to 136-150 kD in Daudi cell membranes. Such variations in the mean value and the range of molecular mass between IFNAR in different cell lines suggest differences in glycosylation. The majority of glycosylation is N-linked, although there may also be a small amount O-linked oligosaccharide. Deglycosylation of IFNAR in Daudi cell membranes results in a 70 kD IFNAR species, indicating that nearly half of the apparent molecular mass of Daudi cell IFNAR is contributed by carbohydrate moieties.

Enzyme immunoassay using native envelope glycoprotein (gp160) for detection of human immunodeficiency virus type 1 antibodies.

An enzyme immunoassay using the purified native gp160 for the detection of human immunodeficiency virus type 1 (HIV-1) antibody was developed. This assay was determined to be highly specific, since (i) 157 serum samples that were confirmed negative by Western blot (immunoblot) (WB) were negative, (ii) 41 serum samples from populations with medical conditions that might cause nonspecific assay reactivity were all negative, and (iii) all 15 serum samples that showed false-positive reactions in one or more commercial HIV-1 screening tests were negative. The assay gave 100% specificity with a randomly selected and unlinked panel of 1,000 serum samples from healthy blood donors. The sensitivity of the assay was assessed by testing 238 samples confirmed as HIV-1 antibody positive by a standardized WB assay. All 238 serum samples (100%) were reactive in the native gp160 assay. In a dilution panel of 14 weakly WB-positive serum samples, 7 samples reacted two-to fivefold more strongly in the gp160 assay than in a virus lysate-based assay; the remaining 7 samples gave comparable reactivities in the two tests. The reactivities of 13 of these 14 serum samples in the gp160 assay were higher than in a commercial enzyme immunoassay that uses a recombinant envelope protein as the antigen. The native gp160 assay was more sensitive to identify seroconversion. In a well-characterized panel of sequential blood samples from a seroconverter, the new assay detected antibodies at least one sample ahead of the other commercial assays tested.