Identification of ColE1 DNA sequences that direct single strand-to-double strand conversion by a phi X174 type mechanism.

A DNA single-strand initiation sequence, named rriA (called rri-1 previously), was detected in the origin region (Hae II fragment E) of the ColE1 plasmid [Nomura, N. & Ray, D. S. (1980) Proc. Natl. Acad. Sci. USA 77, 6566-6570]. Another site, called rriB, has been found on the opposite strand of Hae II fragment C. Both rriA and rriB (i) direct conversion of chimeric M13 phage single-stranded DNA to parental replicative form DNA in vivo by a rifampicin-resistant mechanism that is dependent on the dnaG and dnaB gene products, (ii) provide effector sites of dATP hydrolysis by primosomal protein n', and (iii) require the same primosomal proteins as phi X174 DNA for directing the in vitro conversion that rriA is the DNA sequence that determines the mechanism of lagging strand synthesis of ColE1 DNA and that the mechanism of discontinuous synthesis involves the primosomal proteins utilized in the in vitro conversion of phi X174 single strands to the double-stranded replicative form.     

Comparative in vitro activities of six new fluoroquinolones and other oral antimicrobial agents against Campylobacter pylori.

The in vitro susceptibilities of 56 clinical isolates of Campylobacter pylori to six new fluoroquinolones and other oral antimicrobial agents were determined by an agar dilution technique. Ciprofloxacin was the most active of the fluoroquinolones (MIC for 90% of strains tested [MIC90], 0.05 microgram/ml). Other fluoroquinolones had variable activities, although most isolates were moderately susceptible to fleroxacin (MIC90, 4 micrograms/ml) and lomefloxacin (MIC90, 4 micrograms/ml).     

Activities of daptomycin and teicoplanin against Staphylococcus haemolyticus and Staphylococcus epidermidis, including evaluation of susceptibility testing recommendations.

The in vitro activities of daptomycin, teicoplanin, and three other antimicrobial agents were determined against 105 strains of Staphylococcus haemolyticus and 92 strains of Staphylococcus epidermidis. The MICs for 90% of strains tested (MIC90s) of fusidic acid and rifampin were less than or equal to 0.25 microgram/ml. The MIC90s of daptomycin and vancomycin were less than or equal to 4 micrograms/ml. Teicoplanin had a comparable MIC90 of less than or equal to 4 micrograms/ml for isolates of S. epidermidis. However, MIC90s were 8 and 16 micrograms/ml for oxacillin-susceptible and oxacillin-resistant S. haemolyticus, respectively. Disk diffusion tests were evaluated for daptomycin and teicoplanin. Disks with 30 micrograms of teicoplanin performed satisfactorily when S. epidermidis was tested, but when S. haemolyticus was tested, there was a very major error rate of 10% and a minor error rate of 38%.     

Molecular epidemiological characterization of respiratory isolates of Moraxella catarrhalis in a pediatric intensive care unit

A perceived increase in the number of isolates of Moraxella catarrhalis from the respiratory secretions of patients intubated in the pediatric intensive care unit prompted a review of the clinical profiles of such patients and restriction enzyme analysis of the strains involved. Over two months, of 192 patients admitted to the unit, 154 were intubated. Of the 46 for whom endotracheal tube specimens were submitted to the laboratory, M catarrhalis was isolated in 12. M catarrhalis was not felt to be a significant respiratory pathogen by the attending medical staff in any of the patients from whom it was isolated. In only two patients (17%) could nosocomial acquisition be firmly invoked. Restriction enzyme analysis of the 12 strains ruled out the presence of an epidemic strain. Isolation of M catarrhalis from intubated children does not necessarily imply pathogenicity nor an outbreak situation.     

Diaziquone given as a continuous infusion is an active agent for relapsed adult acute nonlymphocytic leukemia

Diaziquone given as a bolus has not been effective in patients with relapsed or refractory leukemia. Because of in vitro data suggesting enhancement of diaziquone-induced cytotoxicity for human and murine leukemia cells with increased duration of drug exposure and the relatively short terminal plasma half-life of diaziquone, 49 patients (34 acute nonlymphocytic leukemia [ANLL], six chronic myelogenous leukemia in blast crisis [CML-B], five acute lymphocytic leukemia [ALL], four 2 degrees ANLL) with leukemia were given diaziquone as a continuous infusion for seven days. The maximum tolerated dose was 28 mg/m2/d for seven days. The dose-limiting toxicity was the duration of bone marrow aplasia (median, 49 days to greater than 500 PMNs in responders; range, 28 to 101 days). Nonhematologic toxicity was minimal. Responses occurred only in patients with relapsed ANLL, of whom 26 were treated at effective doses. There were six complete responses (CR) (23%) and two partial responses (PR) (8%), although five of eight responders never achieved platelet counts greater than 100,000/microL. Thrombocytopenia in these patients was felt to be a manifestation of diaziquone effect, not persistence of leukemia. The median duration of CR was 195 days (range, 88 to 860+). One patient had active CNS leukemia at the start of treatment and has had a durable (28+ month) CR in both sites of disease. Diaziquone produced prolonged aplasia in patients with secondary ANLL and CML-B (five of ten patients died aplastic), whereas patients with ALL all had regrowth of leukemia and two failed to become aplastic. The lack of significant nonhematologic toxicity and the activity in patients with relapsed ANLL render diaziquone of interest as second-line therapy or consolidation therapy in first remission for patients with ANLL.     

Nursing home staff attitudes towards residents with dementia: strain and satisfaction with work

BACKGROUND: Occupational and resident characteristics affect levels of staff stress and satisfaction in nursing homes, and levels of staff turnover are high. Working with more cognitively impaired residents, especially on day shift, is associated with high levels of stress in nursing home staff. Workload is highly predictive of the experience of burden and job pressure, while these outcomes vary according to whether staff work full- or part-time. AIMS: To investigate the attitudes of nursing home staff towards residents, strain related to dementia care and satisfaction with work and their associations with demographic, occupational and behavioural disturbance in the home. METHODS: A cross-sectional design was adopted, using a self-completion questionnaire survey of 253 nursing home staff from 12 nursing homes in Sydney's eastern suburbs, and behavioural assessment of all 647 residents from 11 of the 12 homes. Staff attitudes and strain were measured using the Swedish Strain in Nursing Care Assessment Scale and satisfaction using the Swedish Satisfaction with Nursing Care and Work Assessment Scale. RESULTS: Staff members' five most prevalent perceptions of residents with dementia were that they are anxious, have little control over their difficult behaviour, are unpredictable, lonely and frightened/vulnerable. The five attributes staff found most difficult to cope with were being aggressive/hostile, having little control over their difficult behaviour, being stubborn/resistive, deliberately difficult, and unpredictable. Although 91% of staff reported that they were happy in their job, a quarter reported that residents provided no job satisfaction. The five satisfaction statements most agreed with were "The patients/residents at work nearly always receive good care", "It is important to try and enter into the way patients experience what happens to them", "Relatives are given enough information about care and treatment", "I enjoy my current work situation" and "Our work organisation is good". There were significant differences between homes in levels of strain related to dementia care that were not accounted for by the level of behavioural disturbance. CONCLUSIONS: Nursing home staff tended to perceive residents in more negative than positive ways. Staff were generally satisfied with their work. Factors other than resident behavioural disturbance are important influences in nursing staff strain.     

Antimicrobial resistance in respiratory tract Streptococcus pneumoniae isolates: results of the Canadian Respiratory Organism Susceptibility Study, 1997 to 2002

A total of 6,991 unique patient isolates of Streptococcus pneumoniae were collected from October 1997 to June 2002 from 25 medical centers in 9 of the 10 Canadian provinces. Among these isolates, 20.2% were penicillin nonsusceptible, with 14.6% being penicillin intermediate (MIC, 0.12 to 1 microg/ml) and 5.6% being penicillin resistant (MIC, > or =2 microg/ml). The proportion of high-level penicillin-resistant S. pneumoniae isolates increased from 2.4 to 13.8% over the last 3 years of the study, and the proportion of multidrug-resistant S. pneumoniae isolates increased from 2.7 to 8.8% over the 5-year period. Resistant rates (intermediate and resistant) among non-beta-lactam agents were as follows: macrolides, 9.6 to 9.9%; clindamycin, 3.8%; doxycycline, 5.5%; chloramphenicol, 3.9%; and trimethoprim-sulfamethoxazole, 19.0%. Rates of resistance to non-beta-lactam agents were higher among penicillin-resistant strains than among penicillin-susceptible strains. No resistance to vancomycin or linezolid was observed; however, 0.1% intermediate resistance to quinupristin-dalfopristin was observed. The rate of macrolide resistance (intermediate and resistant) increased from 7.9 to 11.1% over the 5 years. For the fluoroquinolones, the order of activity based on the MICs at which 50% of isolates are inhibited (MIC(50)s) and the MIC(90)s was gemifloxacin > clinafloxacin > trovafloxacin > moxifloxacin > grepafloxacin > gatifloxacin > levofloxacin > ciprofloxacin. The investigational compounds ABT-773 (MIC(90), 0.008 microg/ml), ABT-492 (MIC(90), 0.015 microg/ml), GAR-936 (tigecycline; MIC(90), 0.06 microg/ml), and BMS284756 (garenoxacin; MIC(90), 0.06 micro g/ml) displayed excellent activities. Despite decreases in the rates of antibiotic consumption in Canada over the 5-year period, the rates of both high-level penicillin-resistant and multidrug-resistant S. pneumoniae isolates are increasing in Canada.     

Anti-graft-versus-host disease effect of DT390-anti-CD3sFv, a single- chain Fv fusion immunotoxin specifically targeting the CD3 epsilon moiety of the T-cell receptor

In a recent study, we showed that an immunotoxin (IT) made with a conventional monoclonal antibody targeting the CD3 epsilon moiety of the T-cell receptor (TCR) had a potent, but partial, graft-versus-host disease (GVHD) effect (Vallera et al, Blood 86:4367, 1995). Therefore, in this current study, we determined whether a fusion immunotoxin made with anti-CD3 single-chain Fv (sFv), the smallest unit of antibody recognizing antigen, would have anti-GVHD activity. A fusion protein was synthesized from a construct made by splicing sFv cDNA from the hybridoma 145-2C11 to a truncated form of the diphtheria toxin (DT390) gene. DT390 encodes a molecule that retains full enzymatic activity, but excludes the native DT binding domain. The DT390-anti-CD3sFv hybrid gene was cloned into a vector under the control of an inducible promoter. The protein was expressed in Escherichia coli and then purified from inclusion bodies. The DT390 moiety of the protein had full enzymatic activity compared with native DT and DT390-anti-CD3sFv, with an IC50 of 1 to 2 nmol/L against phytohemagglutinin-stimulated and alloantigen-stimulated T cells. Specificity was shown (1) by blocking the IT with parental anti-CD3 antibody, but not with a control antibody; (2) by failure of DT390-anti-CD3sFv to inhibit lipopolysaccharide-stimulated murine B cells; (3) by failure of an Ig control fusion protein, DT390-Fc, to inhibit T-cell responses; and (4) with in vivo immunohistochemisty studies. GVHD was studied in a model in which C57BL/6 (H-2b)-purified lymph node T cells were administered to major histocompatibility complex (MHC) antigen disparate unirradiated C.B.-17 scid (H-2d) mice to assess GVHD effects in the absence of irradiation toxicity. Flow cytometry studies showed that donor T cells were expanded 57-fold and histopathologic analysis showed the hallmarks of a lethal model of GVHD. Control mice receiving phosphate-buffered saline showed 17% survival on day 80 after bone marrow transplantation, and mice receiving 2 micrograms DT390-Fc fusion toxin control administered in 2 daily doses for 6 days (days 0 through 5) had a 43% survival rate. In contrast, 86% of mice receiving the same dose of DT390-anti-CD3sFv were survivors on day 80, a significant improvement, although survivors still showed histopathologic signs of GVHD. These findings suggest that new anti-GVHD agents can be genetically engineered and warrant further investigation of fusion proteins for GVHD treatment.     

Transforming growth factor-beta1: differential effects on multiple myeloma versus normal B cells

Interleukin-6 (IL-6), a product of bone marrow stromal cells (BMSCs), is a growth factor for multiple myeloma (MM) cells. Transforming growth factor-beta1 (TGF-beta1) is also produced by BMSCs and can regulate IL- 6 secretion by several tissues, including BMSCs. The present study was designed to characterize in vitro tumor growth regulation by TGF-beta1 in MM. Sorted CD38+CD45RA- MM cells secreted significantly more TGF- beta1 (8.2 +/- 2.0 ng/mL) than peripheral blood mononuclear cells (P < .001), splenic B cells (P < .001), and CD40 ligand (CD40L) pretreated B cells (P < .05). TGF-beta1 secretion by MM-BMMCs (3.8 +/- 0.9 ng/mL) was significantly greater than by N-BMMCs (1.2 +/- 0.1 ng/mL, P < .001). MM-BMSCs also secreted significantly more TGF-beta1 (6.6 +/- 2.5 ng/mL, n = 11) than N-BMSCs (4.4 +/- 0.6 ng/mL, P < .02, n = 10) and N- BMSC lines (3.9 +/- 0.2 ng/mL, P < .02, n = 6). TGF-beta1 secretion was correlated with IL-6 secretion in MM-BMSCs. Anti-TGF-beta1 monoclonal antibody both blocked IL-6 secretion by BMSCs and inhibited the increments in IL-6 secretion by BMSCs induced by MM cell adhesion. Moreover, exogenous TGF-beta1 upregulated IL-6 secretion by MM-BMSCs, normal BMSCs, and CD38+ CD45RA- MM cells, as well as tumor cell proliferation. This is in contrast to the inhibitory effect of TGF- beta1 on proliferation and Ig secretion of normal splenic B cells. Finally, retinoblastoma proteins (pRB) are constitutively phosphorylated in MM cells; TGF-beta1 either did not alter or increased pRB phosphorylation. pRB are dephosphorylated in splenic B cells and phosphorylated in CD40L triggered B cells in contrast to its effects on MM cells, TGF-beta1 decreased phosphorylation of pRB in CD40L treated B cells. These results suggest that TGF-beta1 is produced in MM by both tumor cells and BMSCs, with related tumore cell growth. Moreover, MM cell growth may be enhanced by resistance of tumor cells to the inhibitory effects of TGF-beta1 on normal B-cell proliferation and Ig secretion.