Reactive Oxygen Species Contribute to the Bactericidal Effects of the Fluoroquinolone Moxifloxacin in Streptococcus pneumoniae.

We studied the transcriptomic response of Streptococcus pneumoniae to the fluoroquinolone moxifloxacin at a concentration that inhibits DNA gyrase. Treatment of the wild-type strain R6, at a concentration of 10× the MIC, triggered a response involving 132 genes after 30 min of treatment. Genes from several metabolic pathways involved in the production of pyruvate were upregulated. These included 3 glycolytic enzymes, which ultimately convert fructose 6-phosphate to pyruvate, and 2 enzymes that funnel phosphate sugars into the glycolytic pathway. In addition, acetyl coenzyme A (acetyl-CoA) carboxylase was downregulated, likely leading to an increase in acetyl-CoA. When coupled with an upregulation in formate acetyltransferase, an increase in acetyl-CoA would raise the production of pyruvate. Since pyruvate is converted by pyruvate oxidase (SpxB) into hydrogen peroxide (H2O2), an increase in pyruvate would augment intracellular H2O2. Here, we confirm a 21-fold increase in the production of H2O2 and a 55-fold increase in the amount of hydroxyl radical in cultures treated during 4 h with moxifloxacin. This increase in hydroxyl radical through the Fenton reaction would damage DNA, lipids, and proteins. These reactive oxygen species contributed to the lethality of the drug, a conclusion supported by the observed protective effects of an SpxB deletion. These results support the model whereby fluoroquinolones cause redox alterations. The transcriptional response of S. pneumoniae to moxifloxacin is compared with the response to levofloxacin, an inhibitor of topoisomerase IV. Levofloxacin triggers the transcriptional activation of iron transport genes and also enhances the Fenton reaction.

Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells.

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPß) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPß, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction.

The human Lgl polarity gene, Hugl-2, induces MET and suppresses Snail tumorigenesis.

Lethal giant larvae proteins have key roles in regulating polarity in a variety of cell types and function as tumour suppressors. A transcriptional programme initiated by aberrant Snail expression transforms epithelial cells to potentially aggressive cancer cells. Although progress in defining the molecular determinants of this programme has been made, we have little knowledge as to how the Snail-induced phenotype can be suppressed. In our studies we identified the human lethal giant larvae homologue 2, Hugl-2, (Llgl2/Lgl2) polarity gene as downregulated by Snail. Snail binds E-boxes in the Hugl-2 promoter and represses Hugl-2 expression, whereas removal of the E-boxes releases Hugl-2 from Snail repression. We demonstrate that inducing Hugl-2 in cells with constitutive Snail expression reverses the phenotype including changes in morphology, motility, tumour growth and dissemination in vivo, and expression of epithelial markers. Hugl-2 expression reduced the nuclear localization of Snail and thus binding of Snail to its target promoters. Our results placing Hugl-2 within the Snail network as well as its ability to suppress Snail carcinogenesis identifies Hugl-2 as a target molecule driving cascades, which may have preventative and therapeutic promise to minimize cancer progression.

Outcomes of patients with AML and MDS who relapse or progress after reduced intensity allogeneic hematopoietic cell transplantation.

We describe treatment, outcomes and prognostic factors for patients who relapse following transplantation with a reduced intensity conditioning regimen. Seventy consecutive patients with high-risk myeloid malignancies underwent transplant and 25 (36%) relapsed, a median of 120 days later. The median percentage of bone marrow blasts at relapse was 24, the median donor chimerism was 73% and new karyotypic abnormalities occurred in 8 out of 20 (40%) evaluable patients. Twenty-one patients (84%) received aggressive treatment for relapse, including chemotherapy (60%), second hematopoietic cell transplantation (HCT; 52%) and/or donor lymphocyte infusion (DLI; 12%). Thirteen achieved a complete response (CR) and four remain in CR. Median overall survival (OS) after relapse was 6 months (95% confidence interval=2.7-9.9 months), and actuarial 1 year OS was 24%. Most deaths were due to disease progression (17/20, 85%). We did not observe an advantage for cellular therapy (DLI or second transplant) compared to chemotherapy. Salvage therapy for relapse after reduced intensity HCT is feasible, associated with low treatment-related mortality, and may result in prolonged survival in select patients. Studies exploring the optimal treatment for relapse following reduced intensity HCT are warranted.

Yield improvement for manufacture of alpha-proteinase inhibitor.

BACKGROUND AND OBJECTIVES: The aim of this study was to increase the yield of active alpha(1)-proteinase inhibitor (alpha(1)-PI) from Cohn fraction IV-1 paste during the manufacture of this therapeutic protein and to investigate the molecular mechanism for this yield increase. MATERIALS AND METHODS: Dissolution experiments with IV-1 paste investigated the impact of different variables on the yield of alpha(1)-PI activity. Solutions of IV-1 paste prepared under different conditions were assayed for evidence of protease activity by Western blots of alpha(1)-PI following SDS-PAGE, by azocaseinolytic and amidolytic (S-2288) assays, and by zymography, and for the extent of alpha(1)-PI oligomerization by Western blots following nondenaturing PAGE. RESULTS: Minor modification of the manufacturing process by combining dissolution of IV-1 paste with the subsequent pH adjustment (to 9.25-9.50 with NaOH), achieved by addition of a standard concentration of NaOH to the 10-mm Tris base dissolvent for IV-1 paste, was found to give a highly reproducible 9.4 +/- 0.9% increase in yield of active alpha(1)-PI. Solutions of IV-1 paste prepared with this combined dissolvent contained reduced amounts of low molecular weight fragments of alpha(1)-PI, reduced protease activity, and reduced amounts of oligomers of alpha(1)-PI. Addition of the protease inhibitor leupeptin to the 10-mm Tris base dissolvent for IV-1 paste also caused an increase in the yield of alpha(1)-PI activity. CONCLUSIONS: Dissolution of IV-1 paste in a more alkaline medium gave a significant increase in the yield of active alpha(1)-PI. This yield increase was attributed to a reduction both in protease activity and in the extent of oligomerization of alpha(1)-PI.

Pre-transplant ganciclovir and post transplant high-dose valacyclovir reduce CMV infections after alemtuzumab-based conditioning.

Alemtuzumab (Campath-1H)-based conditioning regimens are effective in preventing GVHD, but are associated with very high rates of cytomegalovirus (CMV) infection, a major limitation to their use. We evaluated 85 patients receiving conditioning with fludarabine 30 mg/m2/day (day -7 to day -3), alemtuzumab 20 mg/day (day -7 to day -3), and melphalan 140 mg/m2 on day -2. The initial patients received post transplant CMV prophylaxis with high-dose acyclovir. A very high incidence of CMV viremia was observed as has been commonly reported after alemtuzumab-based conditioning. Sixty-seven subsequent patients received pre-transplant ganciclovir and high-dose valacyclovir after engraftment. The cumulative incidence of CMV infection in the valacyclovir cohort was 29%. This compared favorably to the cumulative incidence of 53% in patients receiving only acyclovir (P = 0.004) and to literature data. CMV prophylaxis with pre-transplant ganciclovir and high-dose valacyclovir after engraftment appears effective in preventing the excessive incidence of CMV infection after alemtuzumab-based conditioning regimens.

Fludarabine, melphalan, and alemtuzumab conditioning in adults with standard-risk advanced acute myeloid leukemia and myelodysplastic syndrome.

PURPOSE: This prospective phase II study evaluated toxicity, relapse rate, progression-free survival, and overall survival after allogeneic transplantation and conditioning with fludarabine, melphalan, and alemtuzumab in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). PATIENTS AND METHODS: Fifty-two consecutive adults with AML and MDS were enrolled onto the study. Median age was 52 years (range, 17 to 71 years) and the majority of patients had high-risk disease, comorbidities, and/or modest reduction in performance status. Fifty-six percent of patients had unrelated or mismatched related donors. RESULTS: After a median follow-up of 18 months (range, 2 to 34 months), 1-year survival was 48% (95% CI, 34% to 61%), progression-free survival was 38% (95% CI, 25% to 52%), relapse rate was 27% (95% CI, 15% to 40%), and treatment-related mortality was 33% (95% CI, 20% to 46%). The cumulative probability of extensive chronic graft-versus-host disease (GVHD) was only 18% (95% CI, 8% to 40%); extensive chronic GVHD was only observed in recipients of unrelated donor transplants. Performance score and disease status were the major predictors of outcome. High-risk disease (ie, active AML or MDS with > 5% blasts) or even modest decreases in performance status were associated with poor outcomes. Patients with standard-risk leukemia (first or second complete remission) or MDS (< 5% blasts) had excellent outcomes despite unfavorable disease characteristics. CONCLUSION: Fludarabine and melphalan combined with in vivo alemtuzumab is a promising transplantation regimen for patients with AML or MDS and low tumor burden. For patients with active disease, this regimen provides at best modest palliation. Despite a low incidence of GVHD, transplantation is still associated with considerable nonrelapse mortality in patients with decreased performance status.

Long-term follow-up of nonmyeloablative allogeneic stem cell transplantation for renal cell carcinoma: The University of Chicago Experience.

Nonmyeloablative allogeneic stem cell transplantation (NST) has considerable activity in patients with metastatic renal cell carcinoma (RCC), although there are limited long-term follow-up data. Between February 1999 and May 2003, 18 patients with metastatic RCC underwent 19 matched-sibling NSTs after conditioning with fludarabine and cyclophosphamide with tacrolimus and mycophenolate mofetil as post-transplant immunosuppression. Among the four objective responses, all were partial and have relapsed with a median response duration of 609 days (range, 107-926). All responders are alive at a median of 41 months. Median overall survival for the entire cohort was 14 months. There were four early treatment-related deaths and one late treatment-related death. Eight patients died from progressive disease and five (28%) from treatment-related mortality. Stratifying transplant outcome as early death, intermediate (no response, no early death), or response, the combination of pre-treatment anemia and decreased performance status, was associated with adverse outcome (P = 0.015) and reduced survival (HR 5.4, 95% confidence interval of 1.4 to 21, P = 0.007). Responders demonstrated prolonged survival compared to nonresponders (P = 0.002). NST leads to durable responses in a minority of metastatic RCC patients. Appropriate patient selection is paramount. Anemia and decreased performance status may enable risk stratification.

Clinical responses following nonmyeloablative allogeneic stem cell transplantation for renal cell carcinoma are associated with expansion of CD8+ IFN-gamma-producing T cells.

Nonmyeloablative allogeneic stem cell transplantation (NST) is thought to be an immunologic therapy in which donor T cells mediate a graft-versus-tumor effect. We recently reported the clinical outcome of a phase II trial of NST in metastatic renal cell carcinoma (RCC). However, the immune response correlates of clinical activity remain unknown. We now describe the analysis of T-cell subsets and T-cell cytokine-producing potential for those patients evaluable for immune monitoring. The incidence of graft-versus-host disease (GVHD) correlated with clinical outcome, with all responders exhibiting chronic GVHD. Following initial tapering of immunosuppression, an increase in the total numbers of CD8+ T cells but not CD4+ T cells was observed among responders compared to nonresponders. In addition, a greater ratio of CD8+ to CD4+ T cells producing IFN-gamma and IL-2 was seen in clinical responders at the time when clinical responses were first detected (day 180 after transplantation). Our results support the hypothesis that the antitumor effects of NST may be mediated by IFN-gamma-producing CD8+ T cells, and indicate that isolation of putative tumor antigen-specific T cells, ideally, should be pursued around day +180.

Thrombolytic potency of acid-stabilized plasmin: superiority over tissue-type plasminogen activator in an in vitro model of catheter-assisted thrombolysis.

Plasmin, the direct fibrinolytic enzyme, was compared with tissue plasminogen activator (t-PA) in an in vitro thrombolysis model. Plasmin has been prepared in a highly pure form from human plasma and has been stabilized against auto-degradation by low-pH formulation. This acidified formulation of plasmin has been designed to have a low buffering capacity so that it can be directly infused into clots in a stable and latently active form. This low-pH formulation has been shown to be equivalent to a neutral-pH formulation of plasmin in its extent of clot lysis. An in vitro model of catheter-assisted thrombolysis has been devised in which large (12 x 0.6 cm), retracted clots are treated with an intrathrombus thrombolytic agent via a multi-sideport catheter. Plasmin dissolves these plasminogen-deficient clots in a dose-dependent manner and is clearly superior to t-PA. In this model system, t-PA exhibits efficacy only when retracted clots are replenished with plasminogen.

Ten adaptive strategies for family and work balance: advice from successful families.

Despite negative media images and social dynamics insensitive to the lives of many dual-career couples, research shows that these families are largely healthy and thriving. In this study, we investigated the adaptive strategies of middle-class, dual-earner couples (N = 47) with children that are successfully managing family and work. Guided by grounded-theory methodology, analysis of interview data revealed that these successful couples structured their lives around 10 major strategies: Valuing family, striving for partnership, deriving meaning from work, maintaining work boundaries, focusing and producing at work, taking pride in dual earning, prioritizing family fun, living simply, making decisions proactively, and valuing time. Each adaptive strategy is defined and illustrated through the participants' own words. Clinical applications for therapists working with dual-earner couples are offered.

AAMFT Master Series tapes: an analysis of the inclusion of feminist principles into family therapy practice.

Content analysis of 23 American Association for Marriage and Family Therapy Master Series tapes was used to determine how well feminist behaviors have been incorporated into "ideal" family therapy practice. Feminist behaviors were infrequent, being evident in fewer than 3% of time blocks in event sampling and 10 of 39 feminist behaviors of the Feminist Family Therapist Behavior Checklist. These eminent therapists most often dealt with empowerment of male clients and management of power differentials in the therapeutic relationship in a relatively feminist manner, but they tended to hold women responsible for family issues, endorsed traditional rather than egalitarian relationships, and overlooked how the social context affects families. Several of the therapists were blatantly sexist in their treatment of female clients, communicating disrespect of and pathologizing them. The few tapes portraying effective incorporation of feminist principles in family therapy indicate that a handful of behaviors are key to this approach.

Plasmin induces local thrombolysis without causing hemorrhage: a comparison with tissue plasminogen activator in the rabbit.

The direct fibrinolytic enzyme, plasmin, was compared with tissue plasminogen activator (TPA) in rabbit models of local thrombolysis and fibrinolytic hemorrhage. Plasmin was produced by solid-phase urokinase activation of plasminogen and purified on benzamidine Sepharose. Applied as an intra-arterial infusion into the thrombosed abdominal aorta under conditions of unimpeded blood flow, plasmin (4 mg/kg) and TPA (2 mg/kg) achieved equivalent clot dissolution and flow restoration. Using the model of restricted blood flow into the thrombosed aorta, which limits local plasminogen supply, plasmin was superior to TPA in clot lysis and vascular reperfusion. Using similar dosages of plasmin (2 or 4 mg/kg) and TPA (1 or 2 mg/kg) in the earpuncture rebleed model. TPA induced rebleeding in a dose-dependent manner from prior puncture sites in 9 of 10 animals, while none of the 10 animals exposed to plasmin rebled from these sites. These results suggest that plasmin is an effective, unique thrombolytic agent, distinguished from the plasminogen activators in current usage by its striking safety profile.

The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp.

BACKGROUND AND AIMS: Hepatic bile acid homeostasis is regulated by negative feedback inhibition of genes involved in the uptake and synthesis of bile acids. Bile acids down-regulate the rate-limiting gene for bile acid synthesis, cholesterol 7alpha-hydroxylase (cyp7a), via bile acid receptor (fxr) activation of an inhibitory nuclear receptor, shp. We hypothesized that shp would also mediate negative feedback regulation of ntcp, the principal hepatic bile acid transporter. METHODS: Primary rat hepatocytes or transfected HepG2 and Cos cells were treated with retinoids with or without bile acids, and effects on bile acid transport and ntcp and shp gene expression and promoter activity were determined. Gel shift assays were performed using synthetic fxr, rxr, and rar proteins. RESULTS: Bile acid treatment of primary rat hepatocytes prevented retinoid activation of ntcp gene expression and function; this corresponded temporally with shp gene activation. Bile acid-mediated down-regulation occurred via fxr-dependent suppression of the ntcp RXR:RAR response element. Moreover, cotransfected shp directly inhibited retinoid activation of the ntcp promoter. CONCLUSIONS: These studies show negative feedback regulation of ntcp by bile acid-activated fxr via induction of shp. This novel regulatory pathway provides a means for coordinated down-regulation of bile acid import and synthesis, thereby protecting the hepatocyte from bile acid-mediated damage in cholestatic conditions.

A phase II trial of docetaxel for peripheral blood stem cell mobilization for patients with breast cancer and ovarian cancer.

As docetaxel is known to have significant antineoplastic activity against breast and ovarian cancer, we explored its application as a peripheral blood stem cell mobilizing agent in 33 women with stage lll-IV ovarian carcinoma (n = 10) or stage ll-lV breast cancer (n = 23) who were in preparation for high-dose chemotherapy. Eleven patients had bone and/or bone marrow involvement with their disease. The median number of prior regimens received before mobilization was two (range 1-3). The three dose levels administered were 100 mg/m(2), 110 mg/m(2) and 120 mg/m(2). Patients received one dose of docetaxel in the outpatient setting followed by G-CSF (10 microg/kg/day) starting 4 days after docetaxel administration. Leukapheresis commenced when WBC >1.0 x 10(9)/l or when the WBC began to rise after reaching a nadir. Ninety-seven percent of patients began leukapheresis within 7-9 days after receiving docetaxel (range 7-10 days). The collection goal was >/=2 x 10(6) CD34(+) cells/kg. Twenty-seven (82%) patients reached this goal in a median of 2 leukapheresis days (range 1-3). No grade 2-4 nonhematologic toxicities were noted. Thirteen patients (55%) showed a WBC nadir >1.0 x 10(9)/l. None of the patients experienced neutropenic fever or required blood or platelet transfusion support. In conclusion, docetaxel + G-CSF is an effective, well-tolerated regimen for PBPC mobilization which can be safely administered in the outpatient setting with minimal toxicity.

A feminist analysis of self-help bestsellers for improving relationships: a decade review.

Self-help literature is pervasive and influential in the United States. A critical analysis of self-help books would help therapists to determine their utility for the therapeutic process and assist them in making reading recommendations to clients. In this study, a content analysis was conducted of the top 11 relationship self-help books on the New York Times Bestseller List over a period of 10 years (1988-1998) to determine the degree to which these books support a feminist approach to therapy. This study yielded three major findings. First, the number of feminist books, the number of nonfeminist books, and those falling in the middle across four components of feminist family therapy are about equal. However, the second major finding was that the top-selling books are more likely to be nonfeminist than feminist. The third finding is that most best-selling self-help books appear to have become less compatible with a feminist approach to relationships over time. This analysis encourages therapists to think critically about these best-selling books; it will also allow therapists to consider this methodology as a model for critically analyzing other books that they recommend to clients or use in their own professional development.

Mars and Venus: unequal planets.

Self-help books, a pervasive and influential aspect of society, can have a beneficial or detrimental effect on the therapeutic process. This article describes a thematic analysis and feminist critique of the best-selling self-help book, Men are from Mars, Women are from Venus. This analysis revealed that the author's materials are inconsistent with significant family therapy research findings and key principles of feminist theories. His descriptions of each gender and his recommendations for improving relationships serve to endorse and encourage power differentials between women and men.