Efficacy, safety and tolerability of topical terbinafine nail solution in patients with mild-to-moderate toenail onychomycosis: results from three randomized studies using double-blind vehicle-controlled and open-label active-controlled designs.

BACKGROUND: Terbinafine nail solution (TNS) was developed for the treatment of onychomycosis. OBJECTIVE: To assess the efficacy of TNS vs. vehicle and amorolfine 5% nail lacquer. METHODS: Subjects with mild-to-moderate toe onychomycosis (25% to ≤75% nail-involvement, matrix uninvolved) were randomized to receive either TNS or vehicle in two double-blind studies, and to TNS or amorolfine in an active-controlled, open-label study. Primary endpoint was complete cure (no residual clinical involvement and negative mycology) at week 52. Secondary endpoints were mycological cure (negative mycology defined as negative KOH microscopy and negative culture) and clinical effectiveness (≤10% residual-involvement and negative mycology) at week 52. RESULTS: Complete cure was not different between TNS vs. vehicle and amorolfine. Mycological cure was higher with TNS vs. vehicle, as was clinical effectiveness with TNS vs. vehicle, and TNS and amorolfine were not different for secondary efficacy endpoints. Patients achieving mycological cure had a better clinical outcome, and efficacy was improved in subjects with milder disease. Post hoc analysis suggests that nail thickness is an important prognostic factor. Moreover, mycological cure may require 6 months of treatment regimen while complete cure and clinical effectiveness may be achievable only after 10 months. A simulation study suggests that longer treatment duration would have resulted in higher complete cure with TNS vs. vehicle. Study treatments were well-tolerated. CONCLUSION: Primary efficacy objectives were not met in the studies reported herein. Possible reasons for failure to achieve significant outcomes include insufficient length of treatment; stringency of primary endpoint and severity of nail involvement of study population.

Self-association of the single-KH-domain family members Sam68, GRP33, GLD-1, and Qk1: role of the KH domain.

Sam68 is a member of a growing family of proteins that contain a single KH domain embedded in a larger conserved domain of approximately 170 amino acids. Loops 1 and 4 of this KH domain family are longer than the corresponding loops in other KH domains and contain conserved residues. KH domains are protein motifs that are involved in RNA binding and are often present in multiple copies. Here we demonstrate by coimmunoprecipitation studies that Sam68 self-associated and that cellular RNA was required for the association. Deletion studies demonstrated that the Sam68 KH domain loops 1 and 4 were required for self-association. The Sam68 interaction was also observed in Saccharomyces cerevisiae by the two-hybrid system. In situ chemical cross-linking studies in mammalian cells demonstrated that Sam68 oligomerized in vivo. These Sam68 complexes bound homopolymeric RNA and the SH3 domains of p59fyn and phospholipase Cgamma1 in vitro, demonstrating that Sam68 associates with RNA and signaling molecules as a multimer. The formation of the Sam68 complex was inhibited by p59fyn, suggesting that tyrosine phosphorylation regulates Sam68 oligomerization. Other Sam68 family members including Artemia salina GRP33, Caenorhabditis elegans GLD-1, and mouse Qk1 also oligomerized. In addition, Sam68, GRP33, GLD-1, and Qk1 associated with other KH domain proteins such as Bicaudal C. These observations indicate that the single KH domain found in the Sam68 family, in addition to mediating protein-RNA interactions, mediates protein-protein interactions.

Combinatorial gene expression using multiple episomal vectors.

Episomal vectors offer a powerful alternative to integrative recombination for transgene expression in mammalian cells. In this study, various combinations of G protein-coupled receptors (GPCRs) and the G protein subunit G(i2)alpha, were stably expressed from separate episomal vectors in 293-EBNA (293E) cells. Each episome did not adversely affect the others, as gauged by episomal copy number, steady-state mRNA levels and the presence of functional receptors and G protein. Cell lines expressing genes from multiple autonomously replicating vectors were stable just two weeks after transfection, and remained stable in continuous culture for at least 5months. Co-expression of supplementary G(i2)alpha with receptor amplifies the magnitude of signal transduction thereby permitting the development of more sensitive high throughput functional assays. Given these results, combinatorial transfection is the strategy of choice for generating stable cell lines expressing multiple genes for the study of signal-transduction pathways or the evaluation of receptor ligands.

Superantigen-mediated human monocyte-T lymphocyte interactions are associated with an MHC class II-, TCR/CD3-, and CD4-dependent mobilization of calcium in monocytes.

The present study was designed to examine the potential involvement of calcium ions as second messengers in the mediation of the staphylococcal enterotoxin A (SEA)/MHC class II-induced activation of human monocytes. Treatment of monocytes with a monomeric form of SEA failed to induce detectable changes in the level of intracellular calcium in either monocytes or THP-1 cells. However, cross-linking of SEA with biotin-avidin induced a rapid and transient increase in calcium levels in monocytes and in INF-gamma-treated THP-1 cells. This artificial cross-linking system was reproduced by natural physiologic ligands expressed on the surface of T lymphocytes. Delayed, transient, and concentration (cell as well as toxin)-dependent increases in the cytoplasmic level of free calcium in SEA-treated monocytes were observed upon the addition of autologous resting T cells or purified CD4+ cells, but not of CD8+ cells, B cells, or neutrophils. Antibodies against MHC class II Ag, TCR/CD3, and CD4 molecules inhibited the SEA-dependent interaction between monocytes and T cells as indicated by significant decreases in the rise of calcium levels observed in monocytes. Anti-CD8 and anti-class I antibodies did not affect the interaction between the monocytes and the T cells and failed to alter the calcium response. Taken together, these results suggest that the SEA-induced, T cell-dependent calcium mobilization in monocytes requires physical interactions between SEA-MHC class II, TCR/CD3, and CD4 molecules. The ability to mediate a T cell-dependent calcium increase in monocytes was shared by several enterotoxins including staphylococcal enterotoxin B and toxic shock syndrome toxin-1. The characteristics of the SEA-mediated calcium mobilization in monocytes strongly support the hypothesis that this response is an integral part of the signal transducing machinery linked to MHC class II molecules.

Human NK cells express CC chemokine receptors 4 and 8 and respond to thymus and activation-regulated chemokine, macrophage-derived chemokine, and I-309.

NK cells respond to various chemokines, suggesting that they express receptors for these chemokines. In this paper, we show that IL-2-activated NK (IANK) cells express CC chemokine receptor 4 (CCR4) and CCR8, as determined by flow cytometric, immunoblot, and RNase protection assays. Macrophage-derived chemokine (MDC), the ligand for CCR4, induces the phosphorylation of CCR4 within 0.5 min of activating IANK cells with this ligand. This is corroborated with the recruitment of G protein-coupled receptor kinases 2 and 3 and their association with CCR4 in IANK cell membranes. Also, CCR4 is internalized between 5 and 45 min but reappears in the membranes after 60 min of stimulation with MDC. MDC, thymus and activation-regulated chemokine (TARC), and I-309 induce the chemotaxis of IANK cells, an activity that is inhibited upon pretreatment of these cells with pertussis toxin, suggesting that receptors for these chemokines are coupled to pertussis toxin-sensitive G proteins. In the calcium release assay, cross-desensitization experiments showed that TARC completely desensitizes the calcium flux response induced by MDC or I-309, whereas both MDC and I-309 partially desensitize the calcium flux response induced by TARC. These results suggest that TARC utilizes CCR4 and CCR8. Our results are the first to show that IL-2-activated NK cells express CCR4 and CCR8, suggesting that these receptors are not exclusive for Th2 cells.

Expression and regulation of chemokine receptors in human natural killer cells.

Using flow cytometric and RNase protection assays, this study examined the expression of chemokine receptors in nonactivated natural killer (NK) cells and compared this expression with NK cells activated with interleukin (IL)-2, which either adhered to plastic flasks (AD) or did not adhere (NA). None of the NK cell subsets expressed CXCR2, CXCR5, or CCR5. The major differences between these cells include increased expression of CXCR1, CCR1, CCR2, CCR4, CCR8, and CX(3)CR1 in AD when compared to NA or nonactivated NK cells. The chemotactic response to the CXC and CC chemokines correlated with the receptor expression except that all 3 populations responded to GRO-alpha, despite their lack of CXCR2 expression. Pretreatment of these cells with anti-CXCR2 did not inhibit the chemotactic response to GRO-alpha. In addition, nonactivated and NA cells responded to fractalkine, although they lack the expression of CX(3)CR1. This activity was not inhibited by anti-CX(3)CR1. Viral macrophage inflammatory protein (vMIP)-I, I-309, and TARC competed with the binding of (125)I-309 to AD cells with varying affinities. Transforming growth factor (TGF)-beta1 but not any other cytokine or chemokine examined including interferon (IFN)-gamma, MIP-3beta, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC) or I-309, up-regulated the expression of CXCR3 and CXCR4 on NK cell surface. This is correlated with increased chemotaxis of NK cells treated with TGF-beta1 toward stromal cell-derived factor (SDF)-1alpha and interferon-inducible protein-10 (IP-10). Messenger RNA for lymphotactin, RANTES, MIP-1alpha, and MIP-1beta, but not IP-10, monocyte chemotactic protein (MCP)-1, IL-8, or I-309 was expressed in all 3 NK cell subsets. Our results may have implications for the dissemination of NK cells at the sites of tumor growth or viral replication. (Blood. 2001;97:367-375)

Superantigens initiate cognate CD4+ T cell/B cell interactions leading to early activation and proliferation of B cells.

Dimerization or even multimerization of various receptors is commonly required for signal transduction. We report here that clustering of major histocompatibility complex class II molecules in human B cells by biotinylated staphylococcal enterotoxin A (SEA) cross-linked with avidin induces an increase in the level of intracellular calcium. This response was abolished by prior treatment with protein tyrosine kinase (PTK) inhibitors, suggesting that SEA-triggered calcium mobilization in B cells is probably dependent on the activation of PTK. The implication of PTK in SEA-induced early B cell activation was then confirmed by demonstrating that cross-linked SEA induces a significant increase in the level of tyrosine phosphorylation in B cells. The requirement of biotinavidin cross-linking in SEA-induced calcium mobilization in B cells can be fulfilled by the addition CD4+ T cells, suggesting a role for CD4 molecules. Using the murine CD4- T cell hybridoma 3DT, or its derivative I1B3 transfected with human CD4 that both express SEA-specific TCR, we confirmed the CD4 requirement for B cell calcium mobilization and that both specific TCR and CD4 molecules are required in early events of B cell activation induced by SEA. The role of CD4 in SEA-induced B cell proliferation was then investigated. SEA-stimulated B cells proliferated in the presence of CD4+ T cells, whereas no response was observed in the presence of CD8+ T cells. The addition of clone I1B3 CD4+ T cells failed to fulfill the requirement of CD4+ T cells in SEA-induced B cell proliferation, indicating the possible involvement of other CD4+ T cell surface molecules in this response. This issue is currently under investigation.

Physical association of Gi2alpha with interleukin-8 receptors.

Interleukin-8 (IL-8), one of the major mediators of the inflammatory response, belongs to a family of chemokines that includes NAP-2 (neutrophil-activating peptide-2) and Gro-alpha and whose biological activities are directed to a great extent toward neutrophils. Two distinct receptors have been described with overlapping, but not identical, binding affinities for IL-8, NAP-2, and Gro-alpha. This study was designed to examine the intracellular pathways activated upon the occupation of each of the IL-8 receptors (IL-8R). The formation of a physical coupling between IL-8 receptors and the alpha-subunit of heterotrimeric G proteins was tested in neutrophils by examining the presence of the former in anti-Galpha immune precipitates. The addition of IL-8 to a suspension of human neutrophils led to a time-dependent detection of IL-8 in anti-Gi2alpha (raised against amino acids 159-168 (LERIAQSDYI) of Gi2alpha) and anti-Gtalpha (raised against the COOH-terminal 10 amino acids (KENLKDCGLF) of Gtalpha), but not anti-Gq, immunoprecipitates. Similar results were obtained in human 293 cells stably transfected with IL-8RA or IL-8RB. The peptide derived from the COOH-terminal sequence of Gt inhibited the co-immunoprecipitation of IL-8R and Gi observed in response to the anti-Gtalpha and anti-Gi2alpha antibodies. On the other hand, the Gi2alpha peptide only inhibited the immunoprecipitation induced by the anti-Gi2alpha antibody. Peptides derived from Gi1alpha or Gi3alpha had no effect in this assay. The introduction of the anti-Gi2alpha or anti-Gtalpha antibodies or their neutralizing peptides, but not the Gi1alpha or Gi3alpha peptides, into 293 IL-8RA or 293 IL-8RB cells completely blocked the calcium responses obtained upon stimulation with IL-8. These results demonstrate that the occupation of either type of IL-8 receptor leads to a physical coupling to the alpha-subunit of Gi2. In addition, the use of the subunit-specific peptides identified two functionally important but distinct regions of Gialpha, one involved in receptor/Gialpha interaction (KENLKDCGLF) and the other mediating downstream signal transmission (LERIAQSDYI). Finally, the results of this study also validate the use of the transfected 293 cell line as a model for the study of the signal transduction pathway(s) initiated by IL-8.

Diverging signal transduction pathways activated by interleukin 8 (IL-8) and related chemokines in human neutrophils. IL-8 and Gro-alpha differentially stimulate calcium influx through IL-8 receptors A and B.

Interleukin 8 (IL-8) and Gro-alpha are members of the CXC branch of a family of cytokines recently designated the "chemokine" superfamily. Recent evidence indicates that, contrary to previously held beliefs, IL-8 and Gro-alpha may not be perceived equivalently by neutrophils. In this study, we have evaluated the effects of IL-8 and Gro-alpha on the rate of calcium influx in human neutrophils and in 293 cells transfected with type A or type B IL-8 receptors. Of these two chemokines, only Gro-alpha induced an influx of calcium in neutrophils as judged by the sensitivity of the mobilization of calcium to the extracellular calcium chelator EGTA and to the nonselective divalent cation channel inhibitor SK&F 96365, as well as by manganese quenching experiments. IL-8 was similarly without effect on the rate of Mn2+ influx in 293 cells transfected with IL-8 receptor A (IL-8RA) or IL-8RB. On the other hand, Gro-alpha induced an SK&F 96365-sensitive increase of the rate of Mn+2 influx in IL-8RB-, but not in IL-8RA-transfected 293 cells. These results indicate not only that neutrophils respond differently to IL-8 than they do to Gro-alpha but, furthermore, that the consequences of the binding of IL-8 and Gro-alpha to IL-8RB are distinct.

Differential utilization of cyclic ADP-ribose pathway by chemokines to induce the mobilization of intracellular calcium in NK cells.

We show here that cyclic adenosine diphosphate-ribose (cADPR) may be a second messenger for chemokines. Extracts collected from NK cells stimulated with IL-8 for 2 min were incubated with beta-NAD for an additional 2 min (designated as IL-8 extracts). This mixture elevated the mobilization of (Ca(2+))(i) in alpha-toxin permeabilized NK cells. This activity was inhibited upon prior incubation of these cells with ruthenium red but not with heparin. Purified cADPR and not Ins 1,4,5 P(3) desensitized NK cells to the calcium mobilization effect of IL-8 extracts. Further analysis showed that ruthenium red and heparin differentially inhibit RANTES-, SDF-1alpha-, or MDC-induced calcium mobilization in IL-2-activated NK cells. Also, introduction of anti-ryanodine receptor antibody inside streptolysin O-permeabilized NK cells resulted in complete inhibition of MDC, and only partial inhibition of RANTES and SDF-1alpha-induced calcium fluxes in NK cells. Collectively, these results suggest that chemokines may utilize the cADPR/ryanodine receptor pathway as well as the Ins 1,4,5 P(3)/Ins 1,4,5 P(3) receptor signaling pathway to induce the accumulation of calcium in NK cells.

Identification of G-protein binding sites of the human interleukin-8 receptors by functional mapping of the intracellular loops.

Interleukin 8 (IL-8) is considered to be a major mediator of the inflammatory response. Recent evidence indicates that a direct physical association occurs between IL-8 receptors and the alpha subunit of guanine nucleotide regulatory protein (Gi(alpha)2) upon stimulation of human neutrophils by IL-8. In the present study, we identified by site-directed mutagenesis key residues within the three intracellular loops of the IL-8RA receptor involved in the interaction with Gi(alpha)2. We first systematically mutated, in groups of two to four, all the residues in the three intracellular loops of the IL-8 type A receptor to alanine and analyzed the mutant receptors transiently expressed in 293 cells. Four residues in the second intracellular loop (Y136, L137, I139, V140) and one residue in the third intracellular loop (M241) were shown to be crucial for mediating calcium signaling in response to IL-8. Other residues in the second and third intracellular loops were also found to affect IL-8RA-mediated signaling, but to a lesser extent. These effects were not due to lower expression or low IL-8 binding affinities to the mutated receptors. Mutagenesis of the residues in the first intracellular loop had only weak effects on the mobilization of calcium induced by IL-8. We then used a coimmunoprecipitation protocol with anti-Gi(alpha)2 antibodies to determine the involvement of the two regions defined above in Gi(alpha)2 coupling to IL-8 type A receptors. Whereas the anti-Gi(alpha)2 antibodies coimmunoprecipitated IL-8 receptors in the wild-type cells, this interaction was lost in cells expressing mutated receptors that affected intracellular calcium mobilization. The peptides corresponding to the regions of the type A receptor found to be critical for Gi(alpha)2 coupling and induction of intracellular calcium mobilization were next introduced into cells expressing wild-type IL-8RA or IL-8RB to assess their role in coupling Gi(alpha)2 to both IL-8 receptors. The results obtained in the latter experiments suggest that the same regions of the second intracellular loop (Y136, L137, I139, V140) and of the third intracellular loop (M241) are critically involved in the coupling of both IL-8RA and IL-8 RB to Gi(alpha)2 as well as to a downstream effector (or effectors) involved in calcium mobilization.

Evidence for a role for SAM68 in the responses of human neutrophils to ligation of CD32 and to monosodium urate crystals.

SAM68 (Src-associated in mitosis 68 kDa) is a member of the signal transduction of activator RNA novel gene family coding for proteins postulated to be involved in signal transduction and activation of RNA. It has been implicated through its phosphorylation status in the control of the transition from the G(1) to the S phases during mitosis. However, the implication and role of SAM68 in nonproliferative cells are unknown. The present study was initiated to examine the role of SAM68 in the phagocytic responses of the terminally differentiated human neutrophils. The results obtained show that SAM68 is present in human neutrophils and that it is tyrosine phosphorylated in response to stimulation by monosodium urate crystals or by ligation of CD32. Stimulation of neutrophils by these agonists decreases the association of SAM68 with Sepharose-conjugated poly-U beads. Additionally, the amount of immunoprecipitable SAM68 was modulated differentially after stimulation by monosodium urate crystals or by CD32 engagement indicating that the posttranslational modifications and/or protein associations of SAM68 induced by these two agonists differed. The results of this study provide evidence for an involvement of SAM68 in signal transduction by phagocytic agonists in human neutrophils and indicate that SAM68 may play a role in linking the early events of signal transduction to the posttranscriptional modulation of RNA.

Small molecule antagonists of the bradykinin B1 receptor.

Screening Pharmacopeia's encoded combinatorial libraries has led to the identification of potent, selective, competitive antagonists at the bradykinin B1 receptor. Libraries were screened using a displacement assay of [3H]-des-Arglo-kallidin ([3H]-dAK) at IMR-90 cells expressing an endogenous human B1 receptor (Bmax = 20,000 receptors/cell, K(D) = 0.5+/-0.1 nM) or against membranes from 293E cells expressing a recombinant human B1 receptor (Bmax = 8,000 receptors/cell, K(D) = 0.5 +/- 0.3 nM). Compound PS020990, an optimized, representative member from the class of compounds, inhibits specific binding of 3H-dAK at IMR-90 cells with a KI of 6 +/- 1 nM. The compound inhibits dAK-induced phosphatidyl inositol turnover (K(Bapp) = 0.4 +/- 0.2 nM) and calcium mobilization (K(Bapp) = 17 +/- 2 nM) in IMR-90 cells. Compounds from the lead series are inactive at the B2 receptor and are > 1000-fold specific for B1 vs. a variety of other receptors, ion channels and enzymes. PS020990 and other related chemotypes therefore offer an excellent opportunity to explore further the role of B1 receptors in disease models and represent a potential therapeutic avenue.