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The pharmacokinetics and acute toxicity of the new anthra[2, 3-b]furan-3-carboxamide derivative anthrafuran (AF) were studied after intravenous (i.v.), intraperitoneal (i.p.), and peroral administrations (p.o.) to rats. AF blood-plasma concentrations in the animals were determined by HPLC. The total clearance, steady-state distribution volume, and mean residence time of AF were 17.4 mL·min–1·kg–1, 6.6 L/kg, and 6.3 h, respectively. The absolute AF bioavailabilities after i.p. and p.o. administration were 96 and 31%, respectively. The elimination half-life of AF was 5 – 6 h. The LD50 values in the rats after i.v., i.p., and p.o. administration were 17.1, 64.6, and 353.6 mg/kg, respectively. Correlations were found between LD50 or the maximum tolerated dose (MTD) for the studied AF administration pathways and the logarithms of the areas under the curve (AF-concentration— time) or maximum AF concentration and natural times to reach it.

The pharmacokinetics of amphamide, a new semisynthetic antifungal antibiotic, are studied after intravenous injection to rats and rabbits. The amphamide concentration in biological samples was determined by HPLC. Amphamide pharmacokinetics in rabbits were nonlinear. Amphamide levels in animal blood plasma decreased in a triphasic manner. The total clearance and steady-state distribution volume were greater in rats than in rabbits (0.85 vs. 0.3 – 0.5 mL ∙ min–1 ∙ kg–1 and 1.5 vs. 0.6 – 0.9 L/kg, respectively). The mean residence times of amphamide in rats and rabbits were similar (30 vs. 27 – 43 h, respectively). The elimination half-lives calculated using a three-compartment model were 24 h for rats and 31 h for rabbits. The availability of rat tissue for amphamide ranged from 100% (brain) to 1700% (kidneys).

The search for optimal predictors of anti-mutant effects remains a pressing problem in studies of antibiotic-associated bacterial resistance. To relate the emergence of bacterial resistance with the antibiotic mutant prevention concentration (MPC), a novel integral parameter - the area around the resistance threshold, i.e. MPC level (AAMPC) is proposed. The AAMPC is the algebraic sum of the area under the antibiotic concentration-time curve that is above the MPC (positive area) and the area above the concentration-time curve that is under the MPC (negative area). To assess the predictive performance of AAMPC, the enrichment of resistant Staphylococcus aureus was studied by simulating treatment with daptomycin and rifampicin alone and in combination in an in vitro dynamic model. The enhanced anti-mutant effects of the antibiotic combinations were due to lowering the negative 24-h AAMPCs. These findings suggest that a novel MPC-related parameter is a reliable predictor of mutant enrichment.

Current knowledge of the emergence of Streptococcus pneumoniae resistance during treatment with aminopenicillins and macrolides is limited. In particular, clinical reports on isolation of azithromycin-resistant mutants do not relate their enrichment to the actual antibiotic concentrations in blood. In the present work, the selection of amoxicillin- and azithromycin-resistant S. pneumoniae mutants at therapeutic and subtherapeutic antibiotic exposures was studied in an in vitro dynamic model. There was no enrichment of S. pneumoniae mutants resistant to amoxicillin, while azithromycin-resistant mutants were enriched in all simulations. This difference was related to the different times above the mutant prevention concentration: 60-100% of the dosing interval for amoxicillin versus zero percentage for azithromycin. These findings are in concordance with the mutant selection window hypothesis.

To explore the relationship between pharmacokinetic variables and enterococcal resistance to linezolid, a vancomycin-resistant strain whose mutant prevention concentration (MPC) exceeded the MIC by two fold was selected among six clinical isolates of Enterococcus faecium. The selected strain was exposed to simulated pharmacokinetics of twice-daily linezolid for five days. Mutants resistant to 2 × MIC of the antibiotic were enriched at ratios of the 24-h area under the concentration-time curve (AUC24) to the MIC of 15 and 30 h but not at 60 and 120 h. These observations could be explained by the different times when antibiotic concentrations exceed the MPC (T>MPC): 0 to 14, 63 and 100% of the dosing interval. Using the area under the bacterial mutant concentration-time curve (AUBCM) determined in this study and in previous work with other E. faecium strains (MPC/MIC 4), a strain-independent T>MPC relationship with mutant enrichment was established.

The search for optimal predictors of anti-mutant effects remains a pressing problem in studies of antibiotic-associated bacterial resistance. To relate the emergence of bacterial resistance with the antibiotic mutant prevention concentration (MPC), a novel integral parameter - the area around the resistance threshold, i.e. MPC level (AAMPC) is proposed. The AAMPC is the algebraic sum of the area

Current knowledge of the emergence of

To explore if combinations of linezolid (L) with rifampicin (R) are able to restrict Staphylococcus aureus resistance, the enrichment of L- and R-resistant mutants was studied in an in vitro dynamic model. L- and R-resistant mutants were enriched in all single drug treatments. In contrast, L-resistant mutants were not enriched and R-resistant mutants were similar to baseline amounts with only minimal regrowth at the end of the combination treatments. These effects appear to be explained by lowering the mutant prevention concentration (MPC) for L+R combinations (MPCL+R) compared to the MPCs of L and R alone (MPCL and MPCR) and thereby the longer times above MPCL+R (73–100% of the dosing interval for L and 42–58% for R) compared to the times above MPCL (0–44%) and MPCR (0%). These findings provide an opportunity to predict the selection of S. aureus resistance in L+R treatments using MPCL+Rs.

Objectives To test the mutant selection window (MSW) hypothesis applied to linezolid-exposed Staphylococcus aureus and to delineate the concentration-resistance relationship, a mixed inoculum of linezolid-susceptible S. aureus cells and linezolid-resistant mutants (RMs) was exposed to linezolid multiple dosing. Methods Three S. aureus strains (MIC of linezolid 2 mg/L), S. aureus 479, S. aureus 688 and S. aureus ATCC 700699, and their RMs (MIC 8 mg/L) selected by passaging on antibiotic-containing media were used in the study. RMs of S. aureus 479 and S. aureus ATCC 700699 contained a G2576T replacement (Escherichia coli numbering) in one of the copies of the 23S rRNA gene, which had been reported in clinically isolated mutants. Five-day treatments with twice-daily linezolid were simulated over a 32-fold range of the 24 h AUC (AUC24) to the MIC ratio. Results A pronounced enrichment of mutants resistant to 2×, 4× and 8× MIC was observed at AUC24/MIC ratios of 30 and 60 when linezolid concentrations were within the MSW for more than half of the dosing interval for each strain. The number of viable mutant cells decreased significantly at the simulated AUC24/MIC ratio of 120, while the AUC24/MIC ratio of 240 completely prevented mutant enrichment in vitro. Bell-shaped AUBCM-AUC24/MIC and AUBCM-AUC24/MPC relationships (r2 0.91 and 0.79, respectively) were strain independent. Conclusions The bell-shaped pattern of AUC24/MIC and AUC24/MPC relationships with S. aureus resistance to linezolid is consistent with the MSW hypothesis. 'Antimutant' AUC24/MIC ratios were predicted based on the AUC24/MIC relationship with AUBCM.

To predict the effects of combined use of antibiotics on their pharmacodynamics, the susceptibility of Staphylococcus aureus to linezolid-rifampicin combinations was tested at concentration ratios equal to the ratios of 24-area under the concentration-time curve (AUC24) simulated in an in vitro dynamic model. The linezolid MICs in combination with rifampicin decreased 8- to 67-fold. The rifampicin MICs were similar with or without linezolid. The enhanced activity of linezolid combined with rifampicin increased the AUC24/MIC ratios and provided more pronounced antibacterial effects compared with single treatments. The areas between the control growth and time-kill curves (ABBCs) determined in combined and single treatments with linezolid were plotted against AUC24/MIC on the same graph (r2 0.94). These findings suggest that the effects of linezolid-rifampicin combinations can be predicted by AUC24/MICs of linezolid using its MIC determined at pharmacokinetically derived linezolid-to-rifampicin concentration ratios.

In light of the concept of the mutant selection window, i.e., the range between the MIC and the mutant prevention concentration (MPC), MPC-related pharmacokinetic indices should be more predictive of bacterial resistance than the respective MIC-related indices. However, experimental evidence of this hypothesis remains limited and contradictory. To examine the predictive power of the ratios of the area under the curve (AUC 24 ) to the MPC and the MIC, the selection of ciprofloxacin-resistant mutants of four Escherichia coli strains with different MPC/MIC ratios was studied. Each organism was exposed to twice-daily ciprofloxacin for 3 days at AUC 24 /MIC ratios that provide peak antibiotic concentrations close to the MIC, between the MIC and the MPC, and above the MPC. Resistant E. coli was intensively enriched at AUC 24 /MPCs from 1 to 10 h (AUC 24 /MIC from 60 to 360 h) but not at the lower or higher AUC 24 /MPC and AUC 24 /MIC ratios. AUC 24 /MPC and AUC 24 /MIC relationships of the areas under the time courses of ciprofloxacin-resistant E. coli (AUBC M ) were bell-shaped. A Gaussian-like function fits the AUBC M -AUC 24 /MPC and AUBC M -AUC 24 /MIC data combined for all organisms ( r 2 = 0.69 and 0.86, respectively). The predicted anti-mutant AUC 24 /MPC ratio was 58 ± 35 h, and the respective AUC 24 /MIC ratio was 1,080 ± 416 h. Although AUC 24 /MPC was less predictive of strain-independent E. coli resistance than AUC 24 /MIC, the established anti-mutant AUC 24 /MPC ratio was closer to values reported for Staphylococcus aureus (60 to 69 h) than the respective AUC 24 /MIC ratio (1,080 versus 200 to 240 h). This implies that AUC 24 /MPC might be a better interspecies predictor of bacterial resistance than AUC 24 /MIC.

Chemical and mechanical stability of FDTS (perfluorodecyltrichlorosilane) hydrophobic coatings was improved by using an oxide adhesion layer followed by an in-situ vapour deposition of the FDTS self-aligned monolayer. The use of silicon oxide base layer improves stability of the FDTS film and degradation of its hydrophobic properties resulting from a continuous immersion in water and other liquids. We ascribe the improved stability of the FDTS films grown on oxide to high density and uniformity of the surface hydroxyl groups required for FDTS attachment and the resulting high quality of the FDTS monolayer. This approach shows film property improvement over traditional substrates such as silicon and aluminium but may also be particularly useful in biochemistry and micro fluidics when films are deposited on substrates with lower density of the surface hydroxyl groups.

The role of B cells in T-cell priming is unclear, and the effects of B-cell depletion on immune responses to cancer vaccines are unknown. Although results from some mouse models suggest that B cells may inhibit induction of T cell–dependent immunity by competing with antigen-presenting cells for antigens, skewing T helper response toward a T helper 2 profile and/or inducing T-cell tolerance1,2,3,4, results from others suggest that B cells are necessary for priming as well as generation of T-cell memory5,6,7. We assessed immune responses to a well-characterized idiotype vaccine in individuals with severe B-cell depletion but normal T cells after CD20-specific antibody–based chemotherapy of mantle cell lymphoma in first remission. Humoral antigen- and tumor-specific responses were detectable but delayed, and they correlated with peripheral blood B-cell recovery. In contrast, vigorous CD4+ and CD8+ antitumor type I T-cell cytokine responses were induced in most individuals in the absence of circulating B cells. Analysis of relapsing tumors showed no mutations or change in expression of target antigen to explain escape from therapy. These results show that severe B-cell depletion does not impair T-cell priming in humans. Based on these results, it is justifiable to administer vaccines in the setting of B-cell depletion; however, vaccine boosts after B-cell recovery may be necessary for optimal humoral responses.

The clonotypic surface Ig receptor expressed by malignant B cells, idiotype, is a tumor-specific antigen and an attractive target for active immunotherapy. While Ab's specific for tumor idiotype have been well described in patients with B cell malignancies, the precise antigenic epitopes in human idiotype recognized by autologous T cells remain largely unknown. We report here that T cell lines generated from lymphoma patients actively immunized with idiotype protein specifically recognized multiple, unique immunodominant epitopes in autologous tumor idiotype. Synthetic peptides corresponding to hypervariable, but not framework, regions of Ig heavy chain specifically stimulated CD4(+) and CD8(+) T cells to proliferate and secrete proinflammatory cytokines in an MHC-associated manner. Detailed analysis revealed a minimal determinant of an immunodominant epitope, comprising critical residues at the amino terminus that may be a product of somatic hypermutation. Association of idiotype-specific T cell responses with previously documented molecular remissions in idiotype-vaccinated patients suggests that the newly identified T cell epitopes may be clinically relevant. Such antigenic epitopes may serve as candidates for novel peptide-vaccine strategies, and as tools to selectively expand tumor antigen-specific T cells for adoptive immunotherapy and for monitoring T cell immunity in vaccinated patients.

Lymphomas express a tumor-specific antigen which can be targeted by cancer vaccination. We evaluated the ability of a new idiotype protein vaccine formulation to eradicate residual t(14;18)+ lymphoma cells in 20 patients in a homogeneous, chemotherapy-induced first clinical complete remission. All 11 patients with detectable translocations in their primary tumors had cells from the malignant clone detectable in their blood by PCR both at diagnosis and after chemotherapy, despite being in complete remission. However, 8 of 11 patients converted to lacking cells in their blood from the malignant clone detectable by PCR after vaccination and sustained their molecular remissions. Tumor-specific cytotoxic CD8+ and CD4+ T cells were uniformly found (19 of 20 patients), whereas antibodies were detected, but apparently were not required for molecular remission. Vaccination was thus associated with clearance of residual tumor cells from blood and long-term disease-free survival. The demonstration of molecular remissions, analysis of cytotoxic T lymphocytes against autologous tumor targets, and addition of granulocyte-monocyte colony-stimulating factor to the vaccine formulation provide principles relevant to the design of future clinical trials of other cancer vaccines administered in a minimal residual disease setting.

First generation cancer vaccines are being tested for clinical efficacy in the management of metastatic melanoma, colon cancer and non-Hodgkin's lymphoma. Expectations are that more patients will respond to this mode of therapy, and that responders will show longer periods of clinical remission if the potency of these various vaccines can be enhanced. This is being achieved by reformulating these vaccines to immunise the patient optimally for a cytotoxic T-cell and/or antibody response to the tumour-associated antigens. The prevailing strategy for improving the T-cell based vaccines has been to optimise tumour antigen presentation by the HLA molecules. Many of the vaccines designed primarily to elicit an antibody response are immunising against poorly immunogenic carbohydrate antigens. Therefore, increasing their potency through the use of various adjuvants and carrier molecules is being explored. This paper will report on nine therapeutic cancer vaccines currently in clinical trial for melanoma, colon ca...

To delineate the possible advantages of linezolid/doxycycline combinations over either drug alone, the in vitro pharmacodynamics of linezolid, doxycycline and linezolid plus doxycycline were studied with Staphylococcus aureus.S. aureus ATCC 43300 and a clinical isolate S. aureus 479 were exposed to twice-daily linezolid and once-daily doxycycline, alone and in combination, for five consecutive days. Three dosing regimens were simulated with each drug alone: linezolid (AUC(24)/MIC 30, 60 and 200 h-L30, L60 and L200, respectively) and doxycycline (AUC(24)/MIC 90, 180 and 520 h - D90, D180 and D520, respectively) and in combination: linezolid plus doxycycline (L30+D90; L60+D180 and L200+D520).With both S. aureus ATCC 43300 and S. aureus 479 exposed to linezolid or doxycycline, the area between the line crossing each time-kill curve at the level of 10(8) CFU/mL and the respective time-kill curve (I(E)) increased with increasing simulated AUC(24)/MIC ratios. Each of the combined treatments produced greater I(E)s than the sum of linezolid and doxycycline I(E)s observed in the respective single drug treatments.This study suggests that linezolid combinations with doxycycline may be synergistic in treating staphylococcal infections.

The pharmacokinetics of N-(5-oxynicotinoyl)-L-glutamate (ONG) was studied in rats (doses, 20, 100 and 500 mg/kg) and rabbits (50 mg/kg) after bolus administration of calcium salt of N-(5-oxynicotinoyl)-L-glutamic acid (Ampasse preparation). The ONG concentration in the blood serum was determined by HPLC assay with fluorimetric detection. The lower limit of accurate detection for ONG was 100 ng/ml. The ONG pharmacokinetics in rats was linear at relatively small doses (20-100 mg/kg) but nonlinear at a large dose (500 mg/kg). The ONG concentration decay had a two-phase character in both rats and rabbits, so that the pharmacokinetic profiles were fitted to a biexponential equation of the two-compartment model. Systemic pharmacokinetic parameters determined in rats and rabbits, respectively, were as follows: total clearance, 18 and 15 ml/(min kg); steady state distribution volume, 330 and 880 ml/kg; mean retention time, 0.3 and 1.0 h; half-life, 0.73 and 2.3 h. Using the allometric approach to the interspecies extrapolation of the pharmacokinetic data, the half-life of ONG in humans is predicted to be 4 h.

To explore whether the duration of in vitro simulated antibiotic exposure influences bacterial resistance, time-dependent amplification of resistant subpopulations of Staphylococcus aureus was studied in 10 day simulations in a dynamic model with daptomycin as a prototypic agent.S. aureus ATCC 43300 was exposed to once-daily dosing of daptomycin at subtherapeutic ratios of 24 h area under the curve (AUC(24)) to the MIC (32 and 64 h). To provide an integral presentation of the time course of mutants grown on agar plates containing 2x and 4x the MIC of daptomycin, areas under the bacterial mutant kinetic curves (AUBC(M)s) were calculated.Daptomycin-resistant S. aureus mutants were enriched gradually over the entire treatment duration, with systematic increases in AUBC(M) and concomitant decreases in susceptibility. AUBC(M) analyses were also applied to resistance data reported from other studies with S. aureus exposed to daptomycin and garenoxacin over a wide range of AUC(24)/MIC ratios. Although the maximal AUBC(M)s were greater with longer than with shorter exposures, the treatment or observation durations did not influence the predicted anti-mutant AUC(24)/MIC ratios.These findings suggest that the duration of in vitro simulated antibiotic exposure is important for estimates of the maximal enrichment of resistant mutants but not for the prediction of the anti-mutant AUC(24)/MIC ratio.

The results presented in this paper explore the molecular basis for expression of the A48 regulatory Id (RI). A48 RI+ mAb derived from idiotypically manipulated mice molecularly resembled the A48 and UPC 10 prototypes of this system by utilizing a VHX24-Vk10 combination. Id expression by these antibodies was not restricted by a particular D region sequence, JH, or JK segment, but quantitative differences in Id expression were associated with utilization of different members of the VK10 germ-line gene families. The VL sequences of these A48 RI+ mAb has identified amino acid residues lying in four different idiotope-determining regions which may contribute to the structural correlate of this Id. A comparative sequence analysis of the VH regions of these VHX24 utilizing A48 RI+ mAb with several A48 RI+ mAb utilizing VHJ558 or VH7183 VH genes as well as a hybrid transfectoma antibody derived from two A48 RI-, VHJ558 utilizing hybridomas, all suggested that four nonconsecutive positions which lie outside the idiotope-determining regions may contribute structural elements toward expression of this Id. The VH and VL regions of the A48RI+, VHX24-Vk 10+ mAb showed low to moderate levels of somatic mutation which showed different patterns of distribution between the complementary determining region (CDR) and framework regions in the H and L chains. Although the VK sequences contained 50% of the replacement mutations in the CDR, with a replacement/silent mutation ratio of 10, the CDR of the VH sequences contained only 31% of the replacement mutations with a replacement/silent mutation ratio of 0.69.

Wear test polysilicon MEMS microstructures coated with a nanometer-thin layer of carbon-rich alumina exhibit the highest time-to-failure documented to date for hard lubricating coatings.

We propose that a composite hydrophobic SAM film with 100% coverage of all exposed surfaces can be used in many advance packaging applications. For example, a SAM moisture barrier with conventional plastic packaging could be a low cost alternative to hermetic ceramic housed devices and SAMs could be barriers to prevent moisture induced corrosion.

Results on the thermal and immersion stability of ultra-thin composite films created by a deposition method call MVD™ (Molecular Vapor Deposition [1]) are reported. It is observed that these composite films were denser and more stable in thermal and immersion applications when compared to traditional self-assembled monolayer (SAM) films. These improved films were created by a special “sequential” or “layered” deposition process sequence. The MVD™ composite coatings can be deposited at room temperature on a variety of materials such as polymers, fibers, metals, alloys and other materials which normally do not allow films to form with complete surface coverage.Copyright © 2005 by ASME

Purpose: The idiotype (Id) of the immunoglobulin on a given B-cell malignancy is a clonal marker that can serve as a tumor-specific antigen. We developed a novel vaccine formulation by incorporating Id protein with liposomal lymphokine that was more potent than a prototype, carrier-conjugated Id protein vaccine in preclinical studies. In the present study, we evaluated the safety and immunogenicity of this vaccine in follicular lymphoma patients. Experimental Design: Ten patients with advanced-stage follicular lymphoma were treated with five doses of this second generation vaccine after chemotherapy-induced clinical remission. All patients were evaluated for cellular and humoral immune responses. Results: Autologous tumor and Id-specific type I cytokine responses were induced by vaccination in 10 and 9 patients, respectively. Antitumor immune responses were mediated by both CD4+ and CD8+ T cells, were human lymphocyte antigen class I and II associated, and persisted 18 months beyond the completion of vaccination. Specific anti-Id antibody responses were detected in four patients. After a median follow-up of 50 months, 6 of the 10 patients remain in continuous first complete remission. Conclusions: This first clinical report of a liposomal cancer vaccine demonstrates that liposomal delivery is safe, induces sustained tumor-specific CD4+ and CD8+ T-cell responses in lymphoma patients, and may serve as a model for vaccine development against other human cancers and infectious pathogens.

Human germinal center B cell tumors retain the ability of their nontransformed counterparts to somatically hypermutate Ig V genes by nucleotide substitution. Among a survey of 60 primary previously untreated, clonal, follicular lymphomas we have identified a rare VH rearrangement variant and two other in-frame nucleotide insertion/deletion variants within complementarity-determining region III of the Ig heavy chain. The neoplastic origin of the VH rearrangement variant was directly demonstrated in cells isolated by microdissection from malignant follicles. In all three cases a common clonal origin for the variants was demonstrated by complementarity-determining region III nucleotide sequence homology and shared somatic mutations in germline encoded positions in framework region IV. The monoclonal nature of the tumors was independently confirmed by demonstrating a single t(14;18) translocation breakpoint in the two cases with a detectable translocation. All the variants occurred in functional VH rearrangements, which in two cases were directly shown to encode functional Ab molecules. Both recombination-activating genes 1 and 2 were expressed in lymph node tumor cells containing the VH rearrangement variant, although recombination-activating gene expression among a panel of lymphomas was not limited to this variant.

INTRODUCTION Early observations suggesting some cancer patients can mount a therapeutic immune response to their own tumors (reviewed in Ref. 1) have supported long-standing efforts to develop immunotherapy regimens for the adjunct treatment of human neoplastic disease. Immunotherapy protocols are classified as either passive or active specific, depending on how they are designed to deliver the desired immunity to the patient. Passive immunotherapy regimens are characterized by the passive administration of immune effector components such as lymphokines, antibodies, cytotoxic T cells, or lymphocyte-activated-killer (LAK) cells. By contrast, active specific immunotherapy (ASI) regimens actively induce effector mechanisms and tumor immunity in the patient by deliberate vaccination with a tumor cell or its antigenic components. The ASI approach is preferred because the resulting immunity is longer lasting, and a potential memory response may provide beneficial immune surveillance in the host against further ...

Using 334-nm light, we demonstrate side writing of Bragg gratings with an index change of ~10(-4) in germanium-doped fibers. No hydrogen loading of the fibers was required. These gratings have the same temperature stability as gratings fabricated with 240-nm light. Our results suggest that photoionization is not needed for formation of gratings in Ge-doped glass. We observe an enhancement of the 334-nm photosensitivity in boron-codoped fibers and suggest that B facilitates a structural transformation of the glass.

Conditions for deglycosylation of a number of antibiotics belonging to the vancomycin group were studied. A two-stage process including methanolysis followed by acidolysis in a mixture of trifluoracetic acid and HC1 in the presence of nucleophile was shown optimal for formation of a biologically active aglycone of ristomycin A while for formation of the vancomycin aglycone a one-stage process (trifluoracetic acid/HC1--acidolysis) was optimal. A scheme for isolation and purification of the aglycones of ristomycin A and vancomycin is presented.

The study has demonstrated a certain relationship between the content of grisine components with a different length of the polypeptide chain. The relationship allows an assay of the content with respect to the ratio of areas of chromatographic peaks of the short-chained components F and D. This procedure shortens the time spent on an identification of grisine components almost two-fold. It is concluded that the proportion depends on the kinetics of the linear process of the sequential elongation of the component chain.