Pooled analysis of three randomized, double-blind, placebo controlled trials with rimonabant for smoking cessation.

Despite the withdrawal of CB1 antagonists, such as rimonabant, from the market and from active clinical development because of concerns about their side effect profiles, research suggests that the endocannabinoid system may play an important role in modulating nicotine's effects. We report the combined results, using a pooled analysis, of three previously unpublished trials assessing rimonabant as a smoking cessation pharmacotherapy conducted between 2002 and 2004. Smokers (n = 2097) motivated to quit were enrolled in three randomized, double-blind, placebo-controlled trials, STRATUS EU, US, and META, which consisted of a 10-week treatment period with either rimonabant 20 mg (n = 789), 5 mg (n = 518; used in only two of the three studies), or placebo (n = 790), in conjunction with brief counseling. The impact of drug on prolonged abstinence and adverse events was examined at 8 weeks (end-of-treatment) and at 48 weeks (available for STRATUS EU and US) after the targeted quit date. Rimonabant 20 mg resulted in significantly higher abstinence at end-of-treatment and at 48 weeks post-targeted quit date compared with placebo, while rimonabant 5 mg and placebo did not differ. Serious AEs did not differ by drug group. The 20 mg rimonabant dose, compared with placebo, produced increased nausea, diarrhea, anxiety symptoms, hyporexia, and vomiting, and decreased headache, constipation, and cough. These results support rimonabant 20 mg as a modestly effective aid for smoking cessation. Although work on CB1 antagonists such as rimonabant has mostly been stopped because of unacceptable adverse events, these results may inform and spur the development of other endocannabinoids for smoking cessation.

Loss of neutrophil Shp1 produces hemorrhagic and lethal acute lung injury.

The acute respiratory distress syndrome (ARDS) is associated with significant morbidity and mortality and neutrophils are critical to its pathogenesis. Neutrophil activation is closely regulated by inhibitory tyrosine phosphatases including Src homology region 2 domain containing phosphatase-1 (Shp1). Here, we report that loss of neutrophil Shp1 in mice produced hyperinflammation and lethal pulmonary hemorrhage in sterile inflammation and pathogen-induced models of acute lung injury (ALI) through a Syk kinase-dependent mechanism. We observed large intravascular neutrophil clusters, perivascular inflammation, and excessive neutrophil extracellular traps in neutrophil-specific Shp1 knockout mice suggesting an underlying mechanism for the observed pulmonary hemorrhage. Targeted immunomodulation through the administration of a Shp1 activator (SC43) reduced agonist-induced reactive oxygen species in vitro and ameliorated ALI-induced alveolar neutrophilia and NETs in vivo. We propose that the pharmacologic activation of Shp1 has the potential to fine-tune neutrophil hyperinflammation that is central to the pathogenesis of ARDS.

Mitochondrial DNA based phylogeny of the woodpecker genera Colaptes and Piculus, and implications for the history of woodpecker diversification in South America.

The woodpecker genus Colaptes (flickers) has its highest diversity in South America and the closely related genus Piculus is restricted to South and Central America. Two species of flickers occur in North America, and one species is endemic to Cuba. We conducted a Bayesian phylogenetic analysis of three mitochondrial encoded genes (cyt b, COI, 12S rRNA) and confirmed that the two genera are paraphyletic. Three species historically classified as Piculus are actually flickers. We found that the Cuban endemic C. fernandinae is the most basal species within the flickers and that the Northern Flicker is the next most basal species within the Colaptes lineage. The South American clade is most derived. The age of the South American diversification is estimated to be 3.6MY, which is synchronous with the emergence of the Isthmus of Panama. The pattern of diversification of South American flickers is common among South American woodpeckers. Although woodpeckers have their greatest diversity in South America, we hypothesize that woodpeckers (Family Picidae) originated in Eurasia, dispersed to North America via the Bering land bridge, and multiple lineages entered South America as the Isthmus approached its final closing.

Clinician-delivered intervention to facilitate tobacco quitline use by surgical patients.

BACKGROUND: Telephone quitlines that provide counseling support are efficacious in helping cigarette smokers quit and have been widely disseminated; currently, they are underused. Surgery represents a teachable moment for smoking cessation, which can benefit surgical outcomes; however, few surgical patients receive smoking cessation interventions. This study developed and tested a clinician-delivered intervention to facilitate quitline use by adult patients scheduled for elective surgery. METHODS: After formative work involving patients and clinicians, a brief intervention was designed to facilitate telephone quitline use. It was then evaluated in a randomized trial of 300 adults scheduled for elective surgery. A control standard brief stop-smoking intervention served as a comparator, with both interventions delivered by clinicians. The primary outcome was the use rate of a quitline accessed through a dedicated toll-free telephone number, with use defined as completing at least one full counseling session. Secondary outcomes included self-reported abstinence from cigarettes at 30 and 90 days postoperatively. RESULTS: Subject characteristics were similar between the two groups. Records from the designated quitline documented that 29 of 149 subjects (19.5%) in the quitline intervention group and 0 of 151 subjects in the control group completed the first full counseling session (P < 0.0001). There were no significant differences in the self-reported point-prevalent and continuous abstinence rates between groups at either 30 or 90 days postoperatively, although rates tended to be higher in the quitline intervention group. CONCLUSIONS: Clinicians can effectively facilitate quitline use by surgical patients. Further work is necessary to evaluate the efficacy of this approach in terms of long-term abstinence from cigarette smoking.

Lipopolysaccharide (LPS)-induced macrophage activation and signal transduction in the absence of Src-family kinases Hck, Fgr, and Lyn.

Lipopolysaccharide (LPS) stimulates immune responses by interacting with the membrane receptor CD14 to induce the generation of cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and IL-6. The mechanism by which the LPS signal is transduced from the extracellular environment to the nuclear compartment is not well defined. Recently, an increasing amount of evidence suggests that protein tyrosine kinases especially the Src-family kinases Hck, Fgr, and Lyn, play important roles in LPS signaling. To directly address the physiological function of Hck, Fgr and Lyn in LPS signaling, a genetic approach has been used to generate null mutations of all three kinases in a single mouse strain. hck-/-fgr-/-lyn-/- mice are moderately healthy and fertile; macrophages cultured from these mice express normal levels of CD14 and no other Src-family kinases were detected. Although the total protein phosphotyrosine level is greatly reduced in macrophages derived from hck-/-fgr-/-lyn-/- mice, functional analyses indicate that both elicited peritoneal (PEMs) and bone marrow-derived macrophages (BMDMs) from triple mutant mice have no major defects in LPS-induced activation. Nitrite production and cytokine secretion (IL-1, IL-6, and TNF-alpha) are normal or even enhanced in hck-/-fgr-/-lyn-/- macrophages after LPS stimulation. The development of tumor cell cytotoxicity is normal in triple mutant BMDMs and only partially impaired in PEMs after LPS stimulation. Furthermore, the activation of the ERK1/2 and JNK kinases, as well as the transcription factor NF-kappaB, are the same in normal and mutant macrophages after LPS stimulation. The current study provides direct evidence that three Src-family kinases Hck, Fgr, and Lyn are not obligatory for LPS-initiated signal transduction.

Fcgamma receptor-mediated phagocytosis in macrophages lacking the Src family tyrosine kinases Hck, Fgr, and Lyn.

Macrophage Fcgamma receptors (FcgammaRs) mediate the uptake and destruction of antibody-coated viruses, bacteria, and parasites. We examined FcgammaR signaling and phagocytic function in bone marrow-derived macrophages from mutant mice lacking the major Src family kinases expressed in these cells, Hck, Fgr, and Lyn. Many FcgammaR-induced functional responses and signaling events were diminished or delayed in these macrophages, including immunoglobulin (Ig)G-coated erythrocyte phagocytosis, respiratory burst, actin cup formation, and activation of Syk, phosphatidylinositol 3-kinase, and extracellular signal-regulated kinases 1 and 2. Significant reduction of IgG-dependent phagocytosis was not seen in hck(-)(/)-fgr(-)(/)- or lyn(-)(/)- cells, although the single mutant lyn(-)(/)- macrophages did manifest signaling defects. Thus, Src family kinases clearly have roles in two events leading to FcgammaR-mediated phagocytosis, one involving initiation of actin polymerization and the second involving activation of Syk and subsequent internalization. Since FcgammaR-mediated phagocytosis did occur at modest levels in a delayed fashion in triple mutant macrophages, these Src family kinases are not absolutely required for uptake of IgG-opsonized particles.

Independent and opposing roles for Btk and lyn in B and myeloid signaling pathways.

Transphosphorylation by Src family kinases is required for the activation of Bruton's tyrosine kinase (Btk). Differences in the phenotypes of Btk-/- and lyn-/- mice suggest that these kinases may also have independent or opposing functions. B cell development and function were examined in Btk-/-lyn-/- mice to better understand the functional interaction of Btk and Lyn in vivo. The antigen-independent phase of B lymphopoiesis was normal in Btk-/-lyn-/- mice. However, Btk-/-lyn-/- animals had a more severe immunodeficiency than Btk-/- mice. B cell numbers and response to T cell-dependent antigens were reduced. Btk and Lyn therefore play independent or partially redundant roles in the maintenance and function of peripheral B cells. Autoimmunity, hypersensitivity to B cell receptor (BCR) cross-linking, and splenomegaly caused by myeloerythroid hyperplasia were alleviated by Btk deficiency in lyn-/- mice. A transgene expressing Btk at approximately 25% of endogenous levels (Btklo) was crossed onto Btk-/- and Btk-/-lyn-/- backgrounds to demonstrate that Btk is limiting for BCR signaling in the presence but not in the absence of Lyn. These observations indicate that the net outcome of Lyn function in vivo is to inhibit Btk-dependent pathways in B and myeloid cells, and that Btklo mice are a useful sensitized system to identify regulatory components of Btk signaling pathways.

Mapping of the Epstein-Barr virus and C3dg binding sites to a common domain on complement receptor type 2.

Complement receptor type 2 (CR2;CD21), a member of the superfamily of proteins containing short consensus repeats (SCRs), is the B cell receptor for both the gp350/220 envelope protein of Epstein-Barr virus (EBV), and for the C3dg protein of complement. By analysis of CR2 deletion mutants and chimeras formed with CR1 (CD35) we determined that of the 15 SCRs in CR2, the NH2-terminal two SCRs are necessary and sufficient to bind both gp350/220 and C3dg with affinities equivalent to those of the wild-type receptor. The epitope for OKB-7, a mAb that blocks binding of both EBV and C3dg and shares with these ligands B cell-activating capabilities, also requires both SCR-1 and SCR-2, whereas mAbs lacking these functions bind to other SCRs. Thus, EBV, a polyclonal activator of B cells, has selected a site that is proximate or identical to the natural ligand binding site in CR2, perhaps reflecting the relative immutability of that site as well as its signal transducing function.

The molecular mechanism of B cell activation by toll-like receptor protein RP-105.

The B cell-specific transmembrane protein RP-105 belongs to the family of Drosophila toll-like proteins which are likely to trigger innate immune responses in mice and man. Here we demonstrate that the Src-family protein tyrosine kinase Lyn, protein kinase C beta I/II (PKCbetaI/II), and Erk2-specific mitogen-activated protein (MAP) kinase kinase (MEK) are essential and probably functionally connected elements of the RP-105-mediated signaling cascade in B cells. We also find that negative regulation of RP-105-mediated activation of MAP kinases by membrane immunoglobulin may account for the phenomenon of antigen receptor-mediated arrest of RP-105-mediated B cell proliferation.

Negative regulation of phagocytosis in murine macrophages by the Src kinase family member, Fgr.

Ingestion of opsonized pathogens by professional phagocytes results in the generation and release of microbicidal products that are essential for normal host defense. Because these products can result in significant tissue injury, phagocytosis must be regulated to limit damage to the host while allowing for optimal clearance and destruction of opsonized pathogens. To pursue negative regulation of phagocytosis, we assessed the effect of the Src kinase family member, Fgr, on opsonin-dependent phagocytosis by mouse macrophages. We chose Fgr because it is present in high concentrations in circulating phagocytes but is not essential for Fcgamma receptor-mediated ingestion by mouse macrophages. Although expression of Fgr both in a macrophage cell line and in primary macrophages significantly attenuates ingestion mediated by Fcgamma receptors and CR3, it does not affect macropinocytosis or receptor-mediated endocytosis. This selective effect of Fgr is independent of its tyrosine kinase function. After Fcgamma receptor cross-linking, Fgr becomes associated with the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor, SIRPalpha (a member of the signal-regulatory protein family, also known as Src homology 2 domain-containing protein tyrosine phosphatase [SHP] substrate 1 [SHPS-1], brain immunoglobulin-like molecule with tyrosine-based activation motifs [BIT], and P84) and potentiates the association of the phosphatase SHP-1 with SIRPalpha. This association is responsible, at least in part, for decreasing positive signaling essential for optimal phagocytosis. These data demonstrate an important negative regulatory role for this Src kinase family member and suggest that this homeostatic function must be overcome for optimal uptake and clearance of opsonized pathogens.

A critical role for Syk in signal transduction and phagocytosis mediated by Fcgamma receptors on macrophages.

Receptors on macrophages for the Fc region of IgG (FcgammaR) mediate a number of responses important for host immunity. Signaling events necessary for these responses are likely initiated by the activation of Src-family and Syk-family tyrosine kinases after FcgammaR cross-linking. Macrophages derived from Syk-deficient (Syk-) mice were defective in phagocytosis of particles bound by FcgammaRs, as well as in many FcgammaR-induced signaling events, including tyrosine phosphorylation of a number of cellular substrates and activation of MAP kinases. In contrast, Syk- macrophages exhibited normal responses to another potent macrophage stimulus, lipopolysaccharide. Phagocytosis of latex beads and Escherichia coli bacteria was also not affected. Syk- macrophages exhibited formation of polymerized actin structures opposing particles bound to the cells by FcgammaRs (actin cups), but failed to proceed to internalization. Interestingly, inhibitors of phosphatidylinositol 3-kinase also blocked FcgammaR-mediated phagocytosis at this stage. Thus, PI 3-kinase may participate in a Syk-dependent signaling pathway critical for FcgammaR-mediated phagocytosis. Macrophages derived from mice deficient for the three members of the Src-family of kinases expressed in these cells, Hck, Fgr, and Lyn, exhibited poor Syk activation upon FcgammaR engagement, accompanied by a delay in FcgammaR-mediated phagocytosis. These observations demonstrate that Syk is critical for FcgammaR-mediated phagocytosis, as well as for signal transduction in macrophages. Additionally, our findings provide evidence to support a model of sequential tyrosine kinase activation by FcgammaR's analogous to models of signaling by the B and T cell antigen receptors.

Deficiency of Src family kinases p59/61hck and p58c-fgr results in defective adhesion-dependent neutrophil functions.

Cross-linking of the neutrophil-beta 2- or beta 3-related leukocyte response integrins by extracellular matrix (ECM) proteins or monoclonal antibodies (mAb) stimulates cytoskeletal rearrangement leading to cell spreading and respiratory burst. Tyrosin phosphorylation of a variety of proteins and activation of the Src family kinases within polymorphonuclear leukocytes (PMN) have recently been implicated in the intracellular signaling pathways generated by leukocyte integrins (Yan, S.R., L. Fumagalli, and G Berton. 1995. J. Inflammation. 45:217-311.) To directly test whether these functional responses are dependent on the Src family kinases p59/61hck and p58c-fgr, we examined adhesion-dependent respiratory burst in PMNs isolated from hck -/-, fgr -/-, and hck -/- fgr -/- knockout mice. Purified bone marrow PMNS from wild-type mice released significant amounts of O2- when adherent to fibrinogen-, fibronectin-, or collagen-coated surfaces, in the presence of activating agents such as tumor necrosis factor (TNF) or formyl-methionyl-leucyl-phenylalanine, as described for human PMNs. PMNs from hck-/-fgr-/- double-mutant mic, however, failed to respond. This defect was specific for integrin signaling, since respiratory burst was normal in hck-/-fgr-/-PMNs stimulated by immune complexes or PMA. Stimulation of respiratory burst was observed in TNF-primed wild-type PMN plated on surfaces coated with murine intracellular adhesion molecule-1 (ICAM-1), while hck-/-fgr-/- PMNs, failed to respond. Direct cross-linking of the subunits of beta 2 and beta 2 integrins by surface-bound mAbs was elicited O2- production by wild-type PMNs, while the double-mutant hck-/-fgr-/- cells failed to respond. Photomicroscopy and cell adhesion assays revealed that the impaired functional responses of hck-/-fgr-/- PMNs were caused by defective spreading and tight adhesion on either ECM protein- or mAb-coated surfaces. In contrast, hck-/-or fgr-/-single mutant cells produced O2- at levels equivalent to wild-type cells on ECM protein, murine ICAM-1, and antiintegrin mAb-coated surfaces. Hence, either p59/61 hck and p 58c-fgr is required for signaling through leukocyte beta 2 and beta 3 integrins leading to PMN spreading and respiratory burst. This is the first direct genetic evidence of the importance of Src family kinases in integrin signaling within leukocytes, and it is also the best example of overlapping function between members of this gene family within a defined signal transduction pathway.

Precision-to-tolerance capability: an important consideration in tight glucose control.

OBJECTIVE: To assess clinical implications of bias and variance of point-of-care glucometric measurements in cardiac surgery patients with wide variations in postoperative hematocrit. METHODS: Point-of-care glucose measurements were compared with values from laboratory analysis of the same sample of whole blood obtained from cardiac patients early on postoperative days 1 and 2. Twenty nurses collected 89 arterial blood samples from 58 patients during a 4-month period. Bias was measured by using difference scores between paired measurements. Patients were grouped within 5% increments according to hematocrit, and analysis of variance was used to test for differences. Variation was analyzed by precision-to-tolerance analysis within 3 euglycemic tolerance ranges. RESULTS: Laboratory glucose values were 62 to 224 mg/dL; point-of-care measures were 83 to 253 mg/dL. Bias was 10.85 mg/dL across all hematocrit groups. Pairs of laboratory and point-of-care glucose values differed significantly (t(174) = 10.03; P < .001). Bias increased from -2.83 mg/dL for patients with hematocrits exceeding 39% to +16.71 mg/dL for patients with hematocrits between 20% and 24%. The standard deviation of difference scores was 11.59 mg/dL overall. The difference between 5% hematocrit groups was significant (F(4) = 4.11; P = .004). Precision-to-tolerance capability ratios for specification limits of 70 to 300, 90 to 140, and 80 to 110 mg/dL were 0.30, 1.39, and 2.32, respectively. CONCLUSIONS: The direction of bias change between hematocrit groupings was the direction predicted in the manufacturer's information. Precision-to-tolerance measures indicated that the point-of-care equipment was not suitable for testing glucose within the planned "tighter" glycemic standards.

Predictors of smokeless tobacco abstinence.

OBJECTIVES: To investigate predictors of tobacco abstinence among smokeless tobacco (ST) users. METHODS: Logistic regression analyses assessed characteristics associated with tobacco abstinence among ST users receiving bupropion SR. RESULTS: Older age was associated with increased tobacco abstinence in both placebo and bupropion SR groups at end of treatment and one year. Abstinence was lower at one year for subjects with a history of major depression. At end-of-treatment, a 2-way interaction was detected suggesting bupropion SR may be efficacious for subjects with other household tobacco users. CONCLUSIONS: Younger ST users and those with a history of depression are less likely to quit ST use.

Early Targeting of L-Selectin on Leukocytes Promotes Recovery after Spinal Cord Injury, Implicating Novel Mechanisms of Pathogenesis.

L-selectin, a lectin-like receptor on all leukocyte classes, functions in adhesive and signaling roles in the recruitment of myeloid cells from the blood to sites of inflammation. Here, we consider L-selectin as a determinant of neurological recovery in a murine model of spinal cord injury (SCI). Spinal cord-injured, L-selectin knock-out (KO) mice (male) showed improved long-term recovery with greater white matter sparing relative to wild-type (WT) mice and reduced oxidative stress in the injured cord at 72 h post-SCI. There was a partial and transient reduction in accumulation of neutrophils in the injured spinal cords of KOs at 24 h post-injury. To complement these findings with KO mice, we sought a pharmacologic means for lowering L-selectin levels. We found that diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), induced the shedding of L-selectin from the cell surface of myeloid subsets, specifically neutrophils and non-classical monocytes, in the blood and the injured spinal cord. Diclofenac administration to injured WT mice enhanced neurological recovery to a level comparable to that of KOs but did not improve recovery in KOs. While diclofenac treatment had no effect on myeloid cell accumulation, there was a reduction in oxidative stress at 72 h post-SCI. These findings implicate L-selectin in secondary pathogenesis beyond a role in leukocyte recruitment and raise the possibility of repurposing diclofenac for the treatment of SCI.

Defective negative regulation of antigen receptor signaling in Lyn-deficient B lymphocytes.

BACKGROUND: To elucidate the role of the Src family kinase Lyn in B cell receptor (BCR) signaling, we and others previously generated lyn-/- mice and analyzed their B cell responses. Although the initiation of BCR signaling in lyn-/- B cells is delayed, BCR-induced ERK2 activation and proliferation are enhanced. As the co-receptors Fc gamma RIIb1 and CD22 have been shown to be negative regulators of BCR signaling, we have now examined their functional roles in lyn-/- B cells. RESULTS: B cells from lyn-/- mice have increased expression of the protein product of the early response gene egr-1, enhanced activation of Jun N-terminal kinase (JNK), and elevated calcium responses upon BCR cross-linking. Tyrosine phosphorylation of Fc gamma RIIb1 in lyn-/- B cells was reduced but negative regulation of the BCR signal by Fc gamma RIIb1 was only modestly impaired. In contrast, tyrosine phosphorylation of CD22 was greatly decreased in lyn-/- B cells, correlating with the inability of CD22 to downregulate the BCR-induced calcium response in these cells. Surprisingly, CD22 remains capable of regulating the ERK2 and JNK pathways in lyn-/- B cells, which may relate to the small residual increase in BCR-induced CD22 phosphorylation. CONCLUSIONS: BCR signal initiation and negative regulation by Fc gamma RIIb1 is not critically dependent on Lyn. In contrast, Lyn plays a particularly important role in the tyrosine phosphorylation of CD22 and in the consequent inhibition of BCR-induced calcium influx. The net result of the Lyn deficiency in B cells is hyperresponsiveness to antigen stimulation, which may explain the autoimmunity observed in lyn-/- mice.

Lupus-like kidney disease in mice deficient in the Src family tyrosine kinases Lyn and Fyn.

Systemic lupus erythematosus (SLE) is an autoimmune disease whose cause is poorly understood. Mice rendered deficient in specific genes have served as useful animal models in deciphering the genetic control of the disease [1]. We [2] and others [3, 4] previously demonstrated that mice deficient in the Src family tyrosine kinase Lyn developed a mild lupus-like disease with high survival rates. During the course of investigating the functional interaction of Src family kinases, we generated a mouse strain deficient in both Lyn and Fyn. The double-mutant mice died at relatively young ages and developed a severe lupus-like kidney disease. Unlike the double-mutant mice, single mutants deficient in either Lyn or Fyn lived longer and had distinct subsets of the symptoms found in the former. Lyn deficiency led to high levels of autoantibody production and glomerulonephritis, as previously reported [2--4], whereas loss of Fyn contributed to proteinuria by a B and T lymphocyte-independent mechanism. Our data suggest that the severe kidney disease in the double-mutant mice results from a combination of immunological and kidney-intrinsic defects. This new animal model may be informative about the causes of human SLE.

Deficiency of Src family kinases Fgr and Hck results in activation of erythrocyte K/Cl cotransport.

Src-family kinases play a central role in regulation of hematopoietic cell functions. We found that mouse erythrocytes express the Src-family kinases Fgr and Hck, as well as Lyn. To directly test whether Fgr and Hck play any role in erythrocyte function, we analyzed red cells isolated from fgr-/-, hck-/-, and fgr-/- hck-/- knock-out mice. Mean corpuscular hemoglobin concentration and median density are increased, while K content is decreased, in fgr-/- hck-/- double-mutant erythrocytes compared with wild-type, fgr-/-, or hck-/- erythrocytes. Na/K pump and Na/K/Cl cotransport were not altered, but K/Cl cotransport activity was significantly and substantially higher (approximately three-fold) in fgr-/- hck-/- double-mutant erythrocytes. This enhanced K/Cl cotransport activity did not depend on cell age. In fact, in response to bleeding, K/Cl cotransport activity increased in parallel with reticulocytosis in wild-type erythrocytes, while abnormal K/Cl cotransport did not change as a consequence of reticulocytosis in fgr-/- hck-/- double-mutant erythrocytes. Okadaic acid, an inhibitor of a phosphatase that has been implicated in activation of the K/Cl cotransporter, inhibited K/Cl cotransport in wild-type and fgr-/- hck-/- double-mutant erythrocytes to a comparable extent. In contrast, staurosporine, an inhibitor of a kinase that has been suggested to negatively regulate this same phosphatase enhanced K/Cl cotransport in wild-type but not in fgr-/- hck-/- double-mutant erythrocytes. On the basis of these findings, we propose that Fgr and Hck are the kinases involved in the negative regulation of the K/Cl cotransporter-activating phosphatase. Abnormality of erythrocyte K/Cl cotransport in fgr-/- hck-/- double-mutant animals represents the first demonstration that Src-family kinases may be involved in regulation of membrane transport.

Nicotine percentage replacement among smokeless tobacco users with nicotine patch.

To obtain preliminary evidence on the safety and efficacy of high dose nicotine patch therapy among smokeless tobacco (ST) users who consume > or =3 cans of ST per week, we conducted a randomized, placebo-controlled clinical trial with 42 ST users randomized to nicotine patch doses of 21, 42, and 63 mg/day or placebo. Serum nicotine concentrations were measured during ad libitum ST use and nicotine replacement therapy, and percentages of nicotine replacement were calculated. We observed substantial inter-subject variability in nicotine concentrations with ad lib ST use. The mean percentage replacement of ad lib ST use serum nicotine concentrations approximated 100% with the 42 mg/day patch dose (mean+/-S.D., 98.4%+/-45%). Dosing with the 21 mg/day nicotine patch was associated with mean "under-replacement" (53.2%+/-17.1%), and the 63 mg/day nicotine was associated with mean "over-replacement" (159.2%+/-121.9%). We observed symptoms of nausea consistent with nicotine toxicity in two subjects in the 63 mg/day group while no subjects in the 42 mg/day reported these symptoms. We conclude that the use of 42 mg/day nicotine patch therapy is safe and should be considered as initial therapy in the clinical setting among ST users who use > or =3 cans/week.

Bupropion SR for the treatment of smokeless tobacco use.

BACKGROUND: No pharmacotherapies have been shown to increase long-term (> or = 6 months) tobacco abstinence rates among smokeless tobacco (ST) users. Bupropion SR has demonstrated potential efficacy for ST users in pilot studies. We conducted a multicenter, randomized, double-blind, placebo-controlled, clinical trial to assess the efficacy and safety of bupropion SR for tobacco abstinence among ST users. METHODS: Adult ST users were randomized to bupropion SR titrated to 150 mg twice daily (N=113) or placebo (N=112) for 12 weeks plus behavioral intervention. The primary endpoint was the 7-day point-prevalence tobacco abstinence rate at week 12. Secondary outcomes included prolonged and continuous tobacco abstinence rates, craving and nicotine withdrawal, and weight gain. RESULTS: The 7-day point-prevalence tobacco abstinence rates did not differ between bupropion SR and placebo at the end treatment (53.1% versus 46.4%; odds ratio (OR) 1.3; p=0.301). The 7-day point-prevalence abstinence did not differ at weeks 24 and 52. The prolonged and continuous tobacco abstinence rates did not differ at weeks 12, 24, and 52. A time-by-treatment interaction was observed in craving over time with greater decreases in the bupropion SR group. At 12 weeks, the mean (+/-S.D.) weight change from baseline among abstinent subjects was an increase of 1.7 (+/-2.9)kg for the bupropion SR group compared to 3.2 (+/-2.7)kg for placebo (p=0.005). CONCLUSIONS: Bupropion SR did not significantly increase tobacco abstinence rates among ST users, but it significantly decreased craving and weight gain over the treatment period.