Comparing an immediate cessation versus reduction approach to smokeless tobacco cessation.

INTRODUCTION: Relatively few studies have investigated pharmacological or behavioral treatment of smokeless tobacco (ST) users who do not have immediate quit plans. In this study, we compared a reduction treatment approach with an immediate cessation approach in a population of ST users who reported no immediate plans to quit. METHODS: Subjects randomly assigned to the immediate cessation condition set a quit date soon after enrollment and were offered 2 weeks of nicotine patch therapy to help in their cessation efforts. Subjects assigned to the ST reduction group were provided with their choice of either 4 mg nicotine lozenge or ST brand switching to help them reduce their ST use or levels of nicotine exposure, respectively. Quit date was 6 weeks after the onset of treatment. Follow-up was at 12 weeks and 26 weeks postenrollment and 26 weeks postquit. RESULTS: Both 7-day point prevalence abstinence and prolonged abstinence rates following the quit date were significantly higher in the immediate cessation group versus the reduction group at 12 and 26 weeks (all p values ≤ .04) and for prolonged abstinence at 6 months postquit (p = .002). Significant reductions in ST use among nonquitters were observed for both groups (p < .0001) with no differences between groups. CONCLUSION: Our study demonstrated that immediate cessation with an established quit date resulted in greater cessation success than a gradual reduction approach among ST users who do not have an immediate quit plan but are motivated to quit.

Cystic fibrosis transmembrane conductance regulator is involved in airway epithelial wound repair.

The role of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in airway epithelial wound repair was investigated using normal human bronchial epithelial (NHBE) cells and a human airway epithelial cell line (Calu-3) of serous gland origin. Measurements of wound repair were performed using continuous impedance sensing to determine the time course for wound closure. Control experiments showed that the increase in impedance corresponding to cell migration into the wound was blocked by treatment with the actin polymerization inhibitor, cytochalasin D. Time lapse imaging revealed that NHBE and Calu-3 cell wound closure was dependent on cell migration, and that movement occurred as a collective sheet of cells. Selective inhibition of CFTR activity with CFTR(inh)-172 or short hairpin RNA silencing of CFTR expression produced a significant delay in wound repair. The CF cell line UNCCF1T also exhibited significantly slower migration than comparable normal airway epithelial cells. Inhibition of CFTR-dependent anion transport by treatment with CFTR(inh)-172 slowed wound closure to the same extent as silencing CFTR protein expression, indicating that ion transport by CFTR plays a critical role in migration. Moreover, morphologic analysis of migrating cells revealed that CFTR inhibition or silencing significantly reduced lamellipodia protrusion. These findings support the conclusion that CFTR participates in airway epithelial wound repair by a mechanism involving anion transport that is coupled to the regulation of lamellipodia protrusion at the leading edge of the cell.

Secretion of MCP-1 and other paracrine factors in a novel tumor-bone coculture model.

BACKGROUND: The bone-tumor microenvironment encompasses unique interactions between the normal cells of the bone and marrow cavity and the malignant cells from a primary or metastasized cancer. A multitude of paracrine factors within this microenvironment such as the growth factor, TGF-beta, and the chemokine, MCP-1, are secreted by many of these cell types. These factors can act in concert to modulate normal and malignant cell proliferation, malignant cell migration and invasion and, often, mediate bone cancer pain. Although many valuable in vitro and in vivo models exist, identifying the relevant paracrine factors and deciphering their interactions is still a challenge. The aim of our study is to test an ex vivo coculture model that will allow monitoring of the expression, release and regulation of paracrine factors during interactions of an intact femur explant and tumor cells. METHODS: Intact or marrow-depleted neonatal mouse femurs and select murine and human sarcoma or carcinoma cell lines were incubated singly or in coculture in specialized well plates. Viability of the bone and cells was determined by immunohistochemical stains, microscopy and marrow cytopreps. Secretion and mRNA expression of paracrine factors was quantitated by ELISA and real-time RT-PCR. RESULTS: Compartments of the bone were optimally viable for up to 48 h in culture and tumor cells for up to 4 days. Bone was the major contributor of TGF-beta and MMP2 whereas both bone and sarcoma cells secreted the chemokine MCP-1 in cocultures. Synergistic interaction between the femur and sarcoma resulted in enhanced MCP-1 secretion and expression in cocultures and was dependent on the presence of the hematopoietic component of the bone as well as other bone cells. In contrast, coculturing with breast carcinoma cells resulted in reduction of TGF-beta and MCP-1 secretion from the bone. CONCLUSION: These studies illustrate the feasibility of this model to examine paracrine interactions between intact bone and tumor cells. Further study of unique regulation of MCP-1 secretion and signaling between these cell types in different types of cancer will be possible using this simulated microenvironment.

Apical SK potassium channels and Ca2+-dependent anion secretion in endometrial epithelial cells.

Apical uridine triphosphate (UTP) stimulation was shown to increase short circuit current (I(sc)) in immortalized porcine endometrial gland epithelial monolayers. Pretreatment with the bee venom toxin apamin enhanced this response. Voltage-clamp experiments using amphotericin B-permeablized monolayers revealed that the apamin-sensitive current increased immediately after UTP stimulation and was K(+) dependent. The current-voltage relationship was slightly inwardly rectifying with a reversal potential of -52 +/- 2 mV, and the P(K)/P(Na) ratio was 14, indicating high selectivity for K(+). Concentration-response relationships for apamin and dequalinium had IC(50) values of 0.5 nm and 1.8 microm, respectively, consistent with data previously reported for SK3 channels in excitable cells and hepatocytes. Treatment of monolayers with 50 microm BAPTA-AM completely blocked the effects of UTP on K(+) channel activation, indicating that the apamin-sensitive current was also Ca(2+) dependent. Moreover, channel activation was blocked by calmidazolium (IC(50) = 5 microm), suggesting a role for calmodulin in Ca(2+)-dependent regulation of channel activity. RT-PCR experiments demonstrated expression of mRNA for the SK1 and SK3 channels, but not SK2 channels. Treatment of monolayers with 20 nm oestradiol-17beta produced a 2-fold increase in SK3 mRNA, a 2-fold decrease in SK1 mRNA, but no change in GAPDH mRNA expression. This result correlated with a 2.5-fold increase in apamin-sensitive K(+) channel activity in the apical membrane. We speculate that SK channels provide a mechanism for rapidly sensing changes in intracellular Ca(2+) near the apical membrane, evoking immediate hyperpolarization necessary for increasing the driving force for anion efflux following P2Y receptor activation.

Tyrosine phosphatases as regulators of skeletal development and metabolism.

The protein tyrosine kinases (PTK) and the protein tyrosine phosphatases (PTPs) are enzymes which play an integral role in tyrosine phosphorylation-dependent signaling cascades. By catalyzing the phosphorylation and dephosphorylation of cellular proteins, these enzymes direct the steady-state levels of specific phosphoproteins and ultimately dictate the functional state of all cells. The importance of this type of signaling in the skeleton is accepted but poorly understood. The contribution of the PTKs to signaling events in bone has been well studied but, in contrast, the regulation by PTPs is poorly defined. The recent identification of 107 genes within the human genome which encode members of the PTP superfamily emphasizes the need to consider the importance of these proteins in skeletal tissue. In this prospective, we will summarize the present state of our knowledge regarding the function of this enzyme superfamily, illustrating its relevance to the development and maintenance of the skeleton and highlighting future directions that should improve our understanding of these critical signaling molecules.