Incorporating social support into a community-wide smoking-cessation contest

Exposure to foreign particles sometimes causes inflammatory reactions through production of cytokines and chemoattractants by phagocytic cells. In this study, we focused on macrophage migration inhibitory factor (MIF) to evaluate its pathophysiological role in the phagocytic process. Immunohistochemical analysis of human pseudosynovial tissues retrieved at revision of total hip arthroplasty showed that infiltrating mononuclear and multinuclear cells were positively stained by both an anti-CD68 antibody and anti-human MIF antibody. For in vitro study, MIF was released from murine macrophage-like cells (RAW 264.7) in response to phagocytosis of fluorescent-latex beads in a particle dose-dependent manner. Northern blot analysis showed marked elevation of the MIF mRNA level in the phagocytic macrophage-like cells. Moreover, pretreatment of RAW 264.7 cells with rat recombinant MIF increased the extent of phagocytosis by 1.6-fold compared with the control. Taken together, these results suggest that MIF plays an important role by activating macrophages in autocrine and paracrine fashion to phagocytose foreign particles.     

Nonclinical model for assessing gastric effects of bisphosphonates

Mechanisms by which ketones potentiate manganese-bilirubin (Mn-BR)-induced cholestasis are unknown. The purpose of the present study was to investigate the effect of methyl isobutyl ketone (MiBK), a widely used ketonic solvent, at the level of the bile canalicular membrane (BCM) and to verify if altered membrane lipid dynamics could be involved in MiBK-potentiated Mn-BR cholestasis. Male Sprague-Dawley rats were exposed 4 hr/day for 3 days to MiBK vapors (200 or 600 ppm). Eighteen hours after the last exposure, manganese (Mn, 4.5 mg/kg) was given i.v. followed 15 min later by bilirubin (BR, 25 mg/kg). Rats were killed 30 min after BR; liver cell plasma membranes (bile canalicular and sinusoidal), microsomes, mitochondria, and cytosol were isolated by differential centrifugation. Lipids were extracted and cholesterol was measured in each fraction. After Mn-BR and MiBK exposure (600 ppm), results indicated a marked increase in BCM cholesterol content compared to rats exposed to air only. This increase was greater than that due to Mn-BR or MiBK given alone. Also, results indicated that cholesterol increased in a dose-related fashion in BCM after MiBK exposure, whereas PM cholesterol remained unaltered. To identify the source of the increased BCM cholesterol and to permit distinction between de novo cholesterol synthesis and subcellular shifts, the hepatic lipid pool was labeled in vivo with [3H]-cholesterol and [2-14C]-mevalonic acid, a cholesterol synthesis precursor. Results showed that after 600 ppm MiBK exposure, 14C-labeled cholesterol was greater than 3H-labeled cholesterol, indicating that the contribution of de novo cholesterol synthesis to the total cholesterol content of the various isolated hepatocellular fractions was more important than the contribution of intracellular pools. Therefore, increased BCM cholesterol content and enhanced accumulation of newly synthesized cholesterol appear to be involved in MiBK potentiation of Mn-BR-induced cholestasis.     

Use of monoclonal antibodies in diagnosis of paracoccidioidomycosis: new strategies for detection of circulating antigens

The precise diagnosis of paracoccidioidomycosis, in most cases, is established by direct methods and indirect immunological tests. The latter method is reliant on the identification of the host's humoral responses, which are usually impaired or absent in patients with severe juvenile forms of the disease and in immunocompromised patients. Determining disease activity or assessing treatment responses by measuring antibody levels is difficult, since antibody titer may remain elevated or persist at stationary levels, even in the presence of clinical improvement. Consequently, there is a need for alternative tests aimed at the identification of circulating antigens. A modification of the standard hybridoma production method was used to raise a panel of murine monoclonal antibodies (MAbs) against the yeast form of Paracoccidioides brasiliensis. Of these, MAb PIB, directed against an 87-kDa determinant, was used to develop an inhibition ELISA (inh-ELISA) capable of detecting as little as 5.8 ng of circulating antigen per ml of serum. Sera from 46 patients with paracoccidioidomycosis or other mycoses and sera from healthy individuals were evaluated by the inh-ELISA; overall sensitivity was 80.4% (37 of 46 paracoccidioidomycosis patients tested positive), and specificity compared with that of normal controls from areas of endemicity was 81.4%. The inh-ELISA detected circulating antigen in 100% of patients with the acute form of paracoccidioidomycosis and in 83.3 and 60% of patients with the chronic multifocal and unifocal forms of paracoccidioidomycosis according to the patients' clinical presentation. These results indicate that the inh-ELISA with MAb PIB is effective in the detection of circulating antigen and that this test may be useful for monitoring responses to treatment and establishing disease prognoses.     

Effects of changes in blood flow rate on cell death and cell proliferation in carotid arteries of immature rabbits

Spontaneous and experimental changes in arterial blood flow rates affect tissue accumulation in developing arteries. To examine whether cell proliferation and/or cell death are affected by alterations in blood flow, we ligated the left external carotid artery of 3-week-old rabbits, which reduces left common carotid blood flow by 71%. In control arteries and after 2 days of flow reduction, agarose gel electrophoresis of DNA extracted from all carotid arteries resolved multiple low molecular weight bands characteristic of apoptosis; however, DNA fragmentation in arteries carrying reduced blood flow was 2.5-fold higher than that of control arteries. The effect of reduced blood flow on cell death subsequently waned but remained significant at 7 days. Cell death in carotid arteries was also detected by in vivo uptake of propidium iodide, a DNA-binding fluorescent dye that labels the nuclei of nonviable cells. Both smooth muscle and endothelial cells exhibited large and statistically significant increases in labeling index in the flow-reduced artery. Propidium iodide-labeled cells were cleared from the vessel wall within 1 to 4 hours of labeling, and nuclear staining displayed condensation (clumping) of chromatin in all labeled cells at later time points. This time course and nuclear morphology and the rapid clearance of labeled cells are consistent with death via apoptosis. Many propidium iodide-positive cells did not display chromatin condensation immediately after labeling; however, this was also true of cultured endothelial cells that were driven into apoptosis with sphingomyelinase treatment and then double-labeled with propidium iodide and the apoptosis marker annexin V. We infer that propidium iodide can label apoptotic vascular cells before these cells display chromatin condensation that is detectable with fluorescence labeling of DNA. Replication rates of smooth muscle and endothelial cells, determined by 5-bromo-2'-deoxyuridine uptake, were inhibited by >75% with decreased blood flow. The inhibition of proliferation was unabated after 7 days of reduced flow. These findings indicate that the coordinated regulation of cell death and cell proliferation, in response to changes in arterial blood flow rates, contributes to arterial remodeling during development.     

Influence of cell polarity on retrovirus-mediated gene transfer to differentiated human airway epithelia

Gene transfer with recombinant murine leukemia viruses (MuLV) provides the potential to permanently correct inherited lung diseases, such as cystic fibrosis (CF). Several problems prevent the application of MuLV-based recombinant retroviruses to lung gene therapy: (i) the lack of cell proliferation in mature pulmonary epithelia, (ii) inefficient gene transfer with a vector applied to the apical surface, and (iii) low titers of many retroviral preparations. We found that keratinocyte growth factor (KGF) stimulated proliferation of differentiated human tracheal and bronchial epithelia. Approximately 50% of epithelia divided in response to KGF as assessed by bromodeoxyuridine histochemistry. In airway epithelia stimulated to divide with KGF, high-titer ampho- and xenotropic enveloped vectors preferentially infected cells from the basal side. However, treatment with hypotonic shock or EGTA transiently increased transepithelial permeability, enhancing gene transfer with the vector applied to the mucosal surfaces of KGF-stimulated epithelia. Up to 35% of cells expressed the transgene after gene transfer. By using this approach, cells throughout the epithelial sheet, including basal cells, were targeted. Moreover, the Cl- transport defect in differentiated CF airway epithelia was corrected. These findings suggest that barriers to apical infection with MuLV can be overcome.     

Circular dichroism and electron microscopy of a core Y61F mutant of the F1 gene 5 single-stranded DNA-binding protein and theoretical analysis of CD spectra of four Tyr --> Phe substitutions

A core Y61F mutant of the gene 5 single-stranded DNA-binding protein (g5p) of f1 bacterial virus aggregated when expressed from a plasmid, but, after refolding in vitro, it behaved much like wild-type and may be a stability or folding mutant. Circular dichroism (CD) titrations showed the same cooperative polynucleotide binding modes for Y61F and wild-type g5p. There are n = 4 and n congruent with 2.5 modes for binding to poly[d(A)] at low ionic strengths, but n = 4, n = 3, and n congruent with 2-2.5 modes for binding to fd single-stranded viral DNA (fd ssDNA), where n is the number of nucleotides occluded by each bound g5p monomer in a given mode. Y61F g5p has slightly reduced affinity in the n = 4 mode. Electron microscopy showed that Y61F g5p forms left-handed nucleoprotein superhelices indistinguishable from wild-type. Progression from binding to fd ssDNA in the n = 4 to n = 3 to n congruent with 2-2.5 mode is accompanied by an increase in the number of helical turns, an increase from (7.7 +/- 0.3) to (9.5 +/- 0.3) to ( approximately 10-13) g5p dimers per turn, and a decrease in the number of DNA nucleotides per turn. From CD spectra for four of five possible Y --> F g5p mutants, we infer that the fifth tyrosine, Tyr 56, contributes strongly to the CD. Retention of a strong 229 nm CD band in all mutants indicates that all retain elements of the native structure. Spectra of Y26F, Y34F, and Y61F g5p imply limited mobility of the replacement Phe. Comparison of measured with calculated CD spectra also suggests limited mobility for Tyr 26 and Tyr 34 in g5p in solution, and provides new information that the g5p structure in solution may be dominated by Tyr 41 rotamers differing from that stabilized in the crystal.     

A sample purification method for rugged and high-performance DNA sequencing by capillary electrophoresis using replaceable polymer solutions. A. Development of the cleanup protocol

A method for the cleanup of Sanger DNA sequencing reaction products for capillary electrophoresis analysis with replaceable polymer solutions has been developed. A poly(ether sulfone) ultrafiltration membrane pretreated with linear polyacrylamide was first used to remove template DNA from the sequencing samples. Then, gel filtration in a spin column format (two columns per sample) was employed to decrease the concentration of salts below 10 microM in the sample solution. The method was very reproducible and increased the injected amount of the sequencing fragments 10-50-fold compared to traditional cleanup protocols. Using M13mp18 as template, the resulting cleaned-up single DNA sequencing fragments could routinely be separated to more than 1000 bases with a base-calling accuracy of at least 99% for 800 bases. The method is simple and universal and can be easily automated. In the following paper, a systematic study to determine quantitatively the effects of the sample solution components such as high-mobility ions (e.g., chloride and dideoxynucleotides) and template DNA on the injected amount and separation efficiency of the sequencing fragments is presented.     

Molecular analysis of the adaptive response of intestinal bile acid transport after ileal resection in the rat

OBJECTIVES: Major operative trauma like aorta-coronary bypass operation may lead to postoperative immunodisturbance, putting the patient at an increased risk for infection and sepsis. The monocyte/macrophage system and the endotoxin receptor CD14 are important in the early recognition and elimination of invading bacteria. The aim of this study was to analyze changes in membrane-associated CD14 and soluble CD14 during and after cardiac involving cardiopulmonary bypass. METHODS: We studied numbers of leukocytes, monocytes, and monocyte subpopulations, expression of monocyte membrane-associated CD14 and plasma levels of soluble CD14 in 10 patients (63 +/- 8 years of age), who underwent elective cardiopulmonary bypass. RESULTS: Cardiopulmonary bypass induced marked postoperative monocytosis, which was maximal 20 hours after the operation (485 +/- 242 cells/microl before, 1080 +/- 264 cells/microl 20 hours after surgery). Expression of membrane-associated CD14 on classical CD14++ monocytes decreased significantly by 40%, reaching a nadir 20 hours after surgery (p < 0.05). At the time of maximal membrane-associated CD14 suppression, the levels of soluble CD14 measured by enzyme-linked immunosorbent assay were clearly increased (3.2 +/- 1.0 microg/ml before versus 5.6 +/- 1.0 microg/ml 20 hours after, p < 0.001). No significant change of the percentage of small (alpha) and large (beta) forms of soluble CD14 was found. CONCLUSIONS: Cardiopulmonary bypass leads to reduced membrane-associated CD14 expression on peripheral blood monocytes and increased levels of soluble CD14 through shedding or secretion of membrane-associated CD14 from the cell surface. These findings indicate that bypass is associated with significant monocyte activation.     

A phase I-II study of paclitaxel, ifosfamide, and vinorelbine with filgrastim (rhG-CSF) support in advanced non-small-cell lung cancer

PURPOSE: We designed a phase I-II trial of three active agents, paclitaxel, ifosfamide, and vinorelbine, in advanced non-small-cell lung cancer (NSCLC) to: 1) define the dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) of paclitaxel with filgrastim (G-CSF) support; and 2) determine the overall response rate and median survival of patients treated on this regimen. PATIENTS AND METHODS: We treated cohorts of patients with stage IIIB or IV NSCLC with ifosfamide 1.2-1.6 g/m2/day x 3 and vinorelbine 20-25 mg/m2/day x 3 and escalating doses of paclitaxel at 100-175 mg/m2 on day 2 with G-CSF support on a 21-day cycle. One prior experimental single-agent chemotherapy regimen was allowed. RESULTS: Fifty-six patients, were enrolled on this trial: 27 on the phase I portion of the study and an additional 29 at the recommended phase II dose (RPTD). Thirteen patients had received prior chemotherapy. Paclitaxel doses of 175 mg/m2 and 150 mg/m2 produced dose-limiting myelosuppression, and the RPTD was determined to be paclitaxel 135 mg/m2 with ifosfamide 1.2 g/m2/day on days 1-3 and vinorelbine 20 mg/m2/ day on days 1-3 with G-CSF support. The overall response rate was 18%, with a median survival of 6.1 months. Six of 35 patients (17%) treated at the RPTD achieved a partial response to therapy. Grade IV neutropenia was observed in 19 of 35 patients at this dose, with eight patients suffering febrile neutropenia. CONCLUSIONS: This non-cisplatin-containing three-drug regimen has substantial toxicity and low activity in advanced NSCLC, and does not seem to improve on prior regimens. It is unclear whether the lack of efficacy relates to an antagonistic reaction between the specific drugs, administration schedule, or to subtherapeutic doses of the individual agents.     

Quorum sensing in Escherichia coli and Salmonella typhimurium

Escherichia coli and Salmonella typhimurium strains grown in Luria-Bertani medium containing glucose secrete a small soluble heat labile organic molecule that is involved in intercellular communication. The factor is not produced when the strains are grown in Luria-Bertani medium in the absence of glucose. Maximal secretion of the substance occurs in midexponential phase, and the extracellular activity is degraded as the glucose is depleted from the medium or by the onset of stationary phase. Destruction of the signaling molecule in stationary phase indicates that, in contrast to other quorum-sensing systems, quorum sensing in E. coli and S. typhimurium is critical for regulating behavior in the prestationary phase of growth. Our results further suggest that the signaling factor produced by E. coli and S. typhimurium is used to communicate both the cell density and the metabolic potential of the environment. Several laboratory and clinical strains of E. coli and S. typhimurium were screened for production of the signaling molecule, and most strains make it under conditions similar to those shown here for E. coli AB1157 and S. typhimurium LT2. However, we also show that E. coli strain DH5alpha does not make the soluble factor, indicating that this highly domesticated strain has lost the gene(s) or biosynthetic machinery necessary to produce the signaling substance. Implications for the involvement of quorum sensing in pathogenesis are discussed.