Compared with previous reports in prerituximab era, addition of rituximab resulted in reduced PPV and sensitivity of mid- and posttherapy PET in patients with aggressive B-cell NHL.

We examined the effect of zinc on rat neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes as simple heteromers of ␣2, ␣3, or ␣4 and ␤2 or ␤4. Coapplication of zinc with low concentrations of acetylcholine (ՅEC 10 ) resulted in differential effects depending on receptor subunit composition. The ␣2␤2, ␣2␤4, ␣3␤4, ␣4␤2, and ␣4␤4 receptors exhibited biphasic modulation by zinc, with potentiation of the acetylcholine response occurring at 1-100 M zinc and inhibition occurring at higher zinc concentrations. In contrast, ␣3␤2 receptors were only inhibited by zinc (IC 50 ϭ 97 Ϯ 16 M). The greatest potentiating effect of zinc was seen with ␣4␤4 receptors that were potentiated to 560 Ϯ 17% of the response to ACh alone, with an EC 50 of 22 Ϯ 4 M zinc. Cadmium, but not nickel, was also able to potentiate ␣4␤4 receptors. Both zinc potentiation of ␣4␤4 receptors and zinc inhibition of ␣3␤2 receptors were voltage independent. The sensitivity of zinc potentiation of ␣4␤4 to diethylpyrocarbonate treatment and alterations in pH suggested the involvement of histidine residues. Zinc continued to inhibit ␣4␤4 and ␣3␤2 after diethylpyrocarbonate treatment. Application of a potentiating zinc concentration increased the response of ␣4␤2 and ␣4␤4 receptors to saturating ACh concentrations. The rate of Ach-induced desensitization of these receptors was unaffected by zinc. Our results reveal zinc potentiation as a new mode of neuronal nAChR modulation.

We have shown previously that the function of neuronal nicotinic acetylcholine receptors can be modulated by zinc. This modulation varies from potentiation to inhibition, depending on receptor subunit composition and zinc concentration, with the ␣4␤2 and ␣4␤4 receptors displaying the most dramatic potentiation. In this study, we used site-directed mutagenesis to identify glutamate 59 and histidine 162 on the rat ␣4 subunit as potential mediators of zinc potentiation. By modeling the extracellular domain of the receptor pentamer, we locate these residues to two subunit-subunit interfaces that alternate with the two acetylcholine-binding interfaces. Substitution of a cysteine at either position allows additional reduction of zinc potentiation upon treatment with the methanethiosulfonate reagents N-biotinoylaminoethyl methanethiosulfonate (MTSEAbiotin) and [2-(trimethylammonium)ethyl] methanethiosulfonate.Mutagenesis and methanethiosulfonate treatment are most effective at position 162, and the presence of zinc hinders the reaction of MTSEA-biotin with the substituted cysteine at this position, suggesting that ␣4His162 participates in forming a coordination site for zinc. Mutagenesis and methanethiosulfonate treatment are less effective at position 59, suggesting that whereas ␣4Glu59 may be near the zinc coordination site, it may not be participating in coordination of the zinc ion. It is noteworthy that the position of ␣4Glu59 within the neuronal nAChR is identical to that of a residue that lines the benzodiazepinebinding site on GABA A receptors. We suggest that the zinc potentiation sites on neuronal nAChRs are structurally and functionally similar to the benzodiazepine-binding sites on GABA A receptors.

Micromolar zinc potentiates neuronal nicotinic acetylcholine receptors (nAChRs) in a subtype-dependent manner. Zinc potentiates receptor function even at saturating agonist concentrations, without altering the receptor desensitization rate. Potentiation could occur through an increase in the number of available receptors, an increase in single-channel current amplitude, or an increase in single-channel open probability. To distinguish among these possibilities, we examined rat neuronal nAChRs expressed in Xenopus oocytes. Blockade of a large fraction of ACh activated alpha4beta4 or alpha4beta2 receptors by the open channel blocker hexamethonium failed to change the extent of potentiation by zinc, suggesting that zinc does not change the number of available receptors. The single-channel amplitudes of ACh (1 microM) activated alpha4beta4 receptors in outside-out patches were similar in the absence and the presence of 100 microM zinc (3.0 +/- 0.1 and 2.9 +/- 0.1 pA, respectively). To determine the effect of zinc on single-channel open probability, we examined alpha4beta4 receptors in cell-attached patches. The open probability at 100 nM ACh (0.011 +/- 0.002) was increased 4.5-fold by 100 microM zinc (0.050 +/- 0.008), accounting for most of the potentiation observed at the whole cell level. The increase in open probability was due to an increase in burst duration, which increased from 207 +/- 38 ms in the absence of zinc to 830 +/- 189 ms in the presence of zinc. Our results suggest that potentiation of neuronal nAChRs by zinc is due to a stabilization of the bursting states of the receptor.

Inhaled heparin prevents antigen-induced bronchoconstriction and inhibits anti-immunoglobulin E-mediated mast cell degranulation. We hypothesized that the antiallergic action of heparin may be molecular weight dependent. Therefore, we studied the effects of three different low-molecular-weight fractions of heparin [medium-, low-, and ultralow-molecular-weight heparin (MMWH, LMWH, ULMWH, respectively)] on the antigen-induced acute bronchoconstrictor response (ABR) and airway hyperresponsiveness (AHR) in allergic sheep. Specific lung resistance was measured in 22 sheep before and after airway challenge with Ascaris suum antigen, without and after pretreatment with inhaled fractionated heparins at doses of 0.31-5.0 mg/kg. Airway responsiveness was estimated before and 2 h postantigen as the cumulative provocating dose of carbachol in breath units that increased specific lung resistance by 400%. All fractionated heparins caused a dose-dependent inhibition of ABR and AHR. ULMWH was the most effective fraction, with the inhibitory dose causing 50% protection (ID50) against ABR of 0.5 mg/kg, whereas ID50 values of LMWH and MMWH were 1.25 and 1.8 mg/kg, respectively. ULMWH was also the most effective fraction in attenuating AHR; the ID50 values for ULMWH, LMWH, and MMWH were 0.5, 2.5, and 4.7 mg/kg, respectively. These data suggest that 1) fractionated low-molecular-weight heparins attenuate antigen-induced ABR and AHR; 2) there is an inverse relationship between the antiallergic activity of heparin fractions and molecular weight; and 3) ULMWH is the most effective fraction preventing allergic bronchoconstriction and airway hyperresponsiveness.