Preemptive DLI without withdrawal of immunosuppression to promote complete donor T-cell chimerism results in favorable outcomes for high-risk older recipients of alemtuzumab-containing reduced-intensity unrelated donor allogeneic transplant: a prospective phase II trial.

Although pretransplant alemtuzumab can reduce GVHD following allogeneic transplantation, it may also increase the risk of mixed donor T-cell chimerism and infections. We hypothesized that the early use of DLI without withdrawal of immunosuppressive drugs in patients with mixed T-cell chimerism would lower the risk of relapse without significantly increasing the risk of GVHD post DLI. Thirty-six patients (median age 59 years) were treated in this phase II trial using reduced-intensity conditioning including s.c. alemtuzumab (total dose 43 mg) and a PBSC graft from a matched unrelated donor (UD). DLI without withdrawal of immunosuppressive drugs was administered to all 25 patients with <50% donor T-cell chimerism on day +60. The cumulative risks of acute and chronic GVHD were 42% and 59%, respectively. Estimated probabilities of non-relapse mortality (NRM) at day 100 and 1 year were 3% and 14%, respectively. With a median follow up 2.4 years, estimated survivals at day 100, 1 and 2 years were 97%, 71% and 57%, respectively. In multivariate analysis, the occurrence of acute GVHD was associated with an increased risk of mortality, whereas the occurrence of chronic GVHD had a protective effect, associated with decreased relapse and improved disease-free survival. Low-dose alemtuzumab and preemptive DLI provides favorable transplant outcomes including low NRM in an older patient population with high-risk malignancies undergoing UD transplantation.

Advancing the development of diagnostic tests and biomarkers for tuberculosis.

High costs and limited returns on investment have hampered progress in developing new diagnostic tests and treatments for tuberculosis (TB). We need new biomarkers to develop assays that can rapidly, efficiently and reliably detect Mycobacterium tuberculosis infection and disease, identify drug resistance and expedite drug and vaccine development. This can only be accomplished through cross-disciplinary collaborations to facilitate access to human specimens. The Food and Drug Administration, Centers for Disease Control and Prevention, National Institutes of Health, the industry and academia experts came together in a June 2010 workshop to examine the field of TB diagnostic test development and biomarker discovery, identify areas of most urgent need and formulate strategies to address those needs.

Glutamyl-gamma-boronate inhibitors of bacterial Glu-tRNA(Gln) amidotransferase.

Analogues of glutamyl-gamma-boronate (1) were synthesized as mechanism-based inhibitors of bacterial Glu-tRNA(Gln) amidotransferase (Glu-AdT) and were designed to engage a putative catalytic serine nucleophile required for the glutaminase activity of the enzyme. Although 1 provides potent enzyme inhibition, structure-activity studies revealed a narrow range of tolerated chemical changes that maintained activity. Nonetheless, growth inhibition of organisms that require Glu-AdT by the most potent enzyme inhibitors appears to validate mechanism-based inhibitor design of Glu-AdT as an approach to antimicrobial development.

Helicobacter pylori-selective antibacterials based on inhibition of pyrimidine biosynthesis.

We report the discovery of a class of pyrazole-based compounds that are potent inhibitors of the dihydroorotate dehydrogenase of Helicobacter pylori but that do not inhibit the cognate enzymes from Gram-positive bacteria or humans. In culture these compounds inhibit the growth of H. pylori selectively, showing no effect on other Gram-negative or Gram-positive bacteria or human cell lines. These compounds represent the first examples of H. pylori-specific antibacterial agents. Cellular activity within this structural class appears to be due to dihydroorotate dehydrogenase inhibition. Minor structural changes that abrogate in vitro inhibition of the enzyme likewise eliminate cellular activity. Furthermore, the minimum inhibitory concentrations of these compounds increase upon addition of orotate to the culture medium in a concentration-dependent manner, consistent with dihydroorotate dehydrogenase inhibition as the mechanism of cellular inhibition. The data presented here suggest that targeted inhibition of de novo pyrimidine biosynthesis may be a valuable mechanism for the development of antimicrobial agents selective for H. pylori.

Clinical relevance of chest pain during dobutamine stress echocardiography in women.

BACKGROUND: Dobutamine stress echocardiography (DSE) is commonly used for diagnosis and management of patients with known or suspected coronary artery disease. Chest pain occurring during DSE potentially provides additional diagnostic accuracy. Our experience suggests that chest pain occurs frequently in women undergoing DSE. HYPOTHESIS: It was the purpose of this study to determine the frequency with which chest pain occurs in women undergoing DSE and the relation to inducible ischemia or coronary artery stenosis. METHODS: To determine the prevalence and clinical significance or chest pain during DSE, we reviewed the records of 154 consecutive women undergoing DSE in our laboratory. Of these, 59 patients (37.5%) also underwent coronary angiography. The presence or absence of chest pain was correlated with ECG changes, left ventricular wall motion abnormalities during DSE, and coronary stenosis by angiography. RESULTS: Forty-one women (26%) developed chest pain during DSE. Patients experiencing chest pain were older (58.5 +/- 9.3 vs. 54.9 +/- 12.6; p = 0.05), and had lower resting heart rates (71 +/- 12.2 vs. 77.9 +/- 14.9; p = 0.008), but received similar maximum doses of dobutamine and reached comparable peak heart rates (131.1 +/- 17.4 vs. 133.5 +/- 21.7; p = NS). Patients with chest pain more commonly exhibited ST-segment depression > or = 1 mm during dobutamine infusion (13/41, 32%, vs. 17/113, 15%; p = 0.02), but chest pain showed no statistically significant correlation with abnormal DSE or with coronary stenosis. CONCLUSIONS: In women undergoing DSE, chest pain occurs in 26% and does not appear to be related to inducible myocardial ischemia. Electrocardiographic changes occur more frequently in patients who experience chest pain, but are also often unrelated to inducible myocardial ischemia.

Extracellular expression of native human anti-lysozyme fragments in Staphylococcus carnosus.

Xylose-inducible vectors have been constructed for extracellular production of antibody fragments in Staphylococcus carnosus. The pre-pro sequence of S. hyicus lipase was taken as secretional signal sequence, and the S. xylosus Xyl repressor was used to confer xylose inducibility of transcription. Cleavage sites for the IgA protease were engineered between the pre-pro sequence and the antibody fragments to permit removal of the pro sequence. Extracellular expression of the light chain and the Fd fragment of a chimeric Fab fragment containing the variable regions of the anti-lysozyme antibody D1.3 was achieved with these vectors. The pro sequence could be removed from the expression product by IgA protease treatment. When the light chain and the Fd fragment were co-secreted as a protein fusion they accumulated in a structure capable of heterodimerization after IgA cleavage. This fusion contains the pre-pro sequence followed by the light chain, a second IgA site and the Fd fragment.

Using fusions with luxAB from Vibrio harveyi MAV to quantify induction and catabolite repression of the xyl operon in Staphylococcus carnosus TM300.

The luxA,B genes from the Gram-negative marine bacterium Vibrio harveyi MAV were used in Staphylococcus carnosus TM300 as a reporter system for regulated expression of xylose utilization. The luciferase genes were fused to the xyl operon from Staphylococcus xylosus C2a. Expression of bioluminescence was induced through addition of xylose and repressed in the presence of glucose. A method to quantitate bioluminescence directly from the culture is described.

Regulation of Staphylococcus xylosus xylose utilization genes at the molecular level.

We have investigated the regulation of the operon encoding xylose utilization in Staphylococcus xylosus C2a and Staphylococcus carnosus TM300. For in vivo studies, transcriptional fusions of the xylAB regulatory region to the lipase gene from Staphylococcus hyicus were constructed. Repression of lipase activity depended on a functional xylR gene and an xyl operator palindrome downstream of the promoter, while induction was obtained in the presence of xylose. Inactivation of either xylR or the xyl operator led to constitutive expression in the absence of xylose. Crude protein extracts from xylR+ staphylococci led to gel mobility shifts of the xyl regulatory DNA in the absence but not in the presence of xylose. A copper-phenanthroline footprint of the shifted band revealed protection of 28 phosphodiesters from cleavage in each strand of the xyl operator. Thus, the Xyl repressor covers the DNA over more than 2.5 helical turns. Glucose repression of the xyl operon occurs at the level of transcription and is independent of a functional xylR gene. A potential cis-active sequence element for glucose repression is discussed on the basis of sequence similarities to respective elements from bacilli.

The effect of fitting procedure on hearing protector attenuation.

Realistically evaluating the effectiveness of hearing protectors continues to be a major problem in hearing conservation. The purpose of this study was to examine a laboratory-based fitting procedure (User Fit) that was designed to yield hearing protector attenuation values similar to that derived from field studies. Ten subjects who were naive to hearing protectors were used in a repeated measures design that measured real ear attenuation at threshold for two types of plugs. Each subject was tested in two fitting conditions that varied based on the type and degree of assistance given to the subjects by the experimenter. The results showed significant differences in attenuation based on the fitting procedure used, with the User Fit best approximating field data. In addition, a generalized learning effect was noted. The results suggest that any experience with earplugs leads to subsequent improvement in attenuation despite the type of earplug used. Further testing is planned with greater numbers of subjects and additional types of hearing protectors.

Effects of isokinetic soreness-inducing exercise on blood levels of C-reactive protein and creatine kinase.

This study examined whether acute inflammation was the mechanism underlying delayed muscle soreness (DMS) by assessing the effect of soreness-inducing exercise on blood levels of C-reactive protein (CRP), an acute inflammation marker. Sixteen female college students (= 20.6 +/- 2.6 years) performed three sets of 35 isokinetic contractions of the knee flexors and extensors at 120 degrees /set on a Biodex isokinetic dynamometer. Group 1 (N = 8) exercised eccentrically and Group 2 (N = 8) concentrically at an intensity of 80% of a concentric 120 degrees /set peak torque. Pre-exercise and 1, 24, 48 and 72 hours postexercise, DMS of the quadriceps femoris (QF) and hamstrings (HA) were assessed and blood samples were collected for creatine kinase (CK), an indicator of muscle damage, and CRP, which was measured by a radial immunodiffusion procedure. The mean CK values 72 hours postexercise were 14,856 and 360 IU/L for groups 1 and 2, respectively. No significant elevations of CRP occurred in either group. ANOVAs using a split plot factorial design found Group 1 to have significantly larger logarithmic CK elevations, ranked QF soreness, and ranked HA soreness than Group 2. In contrast to myocardial infarct patients and marathon runner investigations, this study did not demonstrate abnormal elevations of CRP when increases in CK were induced. With high-repetition submaximal isokinetic exercise, eccentric contractions induce higher levels of muscle damage and DMS than concentric contractions. Further, the hamstrings are more susceptible to DMS than the quadriceps femoris when eccentric isokinetic exercise is performed at the same relative intensity. J Orthop Sports Phys Ther 1992;16(5):208-214.

Organization, promoter analysis and transcriptional regulation of the Staphylococcus xylosus xylose utilization operon.

The Staphylococcus xylosus xyl genes were cloned in Staphylococcus carnosus by complementation to xylose utilization. Xylose isomerase assays under inducing (xylose present) and non-inducing (xylose absent) conditions indicated the presence of a regulated xylA gene on the recombinant plasmid. The nucleotide sequence (4520 bases) revealed three open reading frames with the same polarity. They were identified by sequence homologies as xylR, encoding the Xyl repressor, xylA, encoding xylose isomerase and xylB, encoding xylulokinase. Primer extension analyses indicated constitutive transcription of xylR and xylose-inducible transcription of xylA. Promoter consensus sequences were found upstream of both transcriptional start sites. A transcriptional terminator between xylR and xylA separates the different transcriptional units. Potential regulatory elements were identified by sequence analysis and suggest a repressor-operator mechanism for the regulation of xylAB expression.

Quantitative analysis of Tn10 Tet repressor binding to a complete set of tet operator mutants.

A saturating oligonucleotide-directed mutagenesis of both tet operators in the tet regulatory sequence was performed yielding mutants with four identical base pair exchanges at equivalent positions in the four tet operator half sides. The mutants were cloned between bipolar lacZ and galK indicator genes on a multicopy plasmid allowing the quantitative analysis of their effects in vivo. In the absence of Tet repressor the mutations lead to considerably different expression levels of both genes. They are discussed with respect to the promoter consensus sequences. In particular, the -10 region of the in vivo active tetPR2 promoter is unambiguously defined by these results. In the presence of Tet repressor most of the mutants exhibit a lower affinity for that protein as determined quantitatively by their reduced expression levels. In general, tet operator recognition is most strongly affected by alterations of base pairs near the center of the palindromic sequence. The most important position is the third base pair, followed by base pairs two, four, five and six, the latter showing similar effects as base pair one. At each position, the four possible base pairs show different affinities for Tet repressor. They are discussed according to their exposure of H-bond donors and -acceptors in the major and minor grooves of the B-DNA. The results are in agreement with major groove contacts at positions two, three and five. At position four a low potential correlation of efficiencies with the H-bonding in the minor groove is found, while mutations at position six seem to influence repressor binding by other mechanisms.