Flea (Ctenocephalides felis) control efficacy of topical indoxacarb on dogs subsequently bathed with a chlorhexidine-ketoconazole shampoo.

OBJECTIVE: An evaluation of the effect of chlorhexidine/ketoconazole shampoo baths on the flea control efficacy of indoxacarb applied topically to dogs. METHODS AND RESULTS: We randomly allocated 18 healthy mixed-breed dogs to 3 groups: shampoo only; indoxacarb treated and medicated shampoo; and indoxacarb treated but not shampooed. Indoxacarb was administered on day 0 and dogs were shampooed on days 9 and 23. Dogs were infested with 100 adult Ctenocephalides felis initially 2 days before treatment and then weekly from days 7 to 28. Fleas were removed and counted 48 h post-infestation. CONCLUSION: Medicated shampoo use did not significantly reduce indoxacarb efficacy against C. felis.

Pharmacokinetic-pharmacodynamic integration of orbifloxacin in Japanese quail (Coturnix japonica) following oral and intravenous administration.

The pharmacokinetics of single-dose administration of orbifloxacin were determined in Japanese quail (Coturnix japonica) at dosages of 5 mg/kg intravenous (i.v. n = 12) and 7.5 mg/kg oral (p.o.; n = 5), 10 mg/kg p.o. (n = 5), 15 mg/kg p.o. (n = 12) and 20 mg/kg p.o. (n = 5) via HPLC. Orbifloxacin minimal inhibitory concentrations (MICs) against 22 microbial isolates from various bird species were performed to calculate pharmacodynamic surrogate markers. The concentration-time data were analyzed using a naïve pooled data (NPD) approach and compartmental and noncompartmental methods. Steady-state volume of distribution (Vd(ss)) and total body clearance (Cl) after i.v. administration were estimated to be 1.27 L/kg and 0.60 L/h·kg, respectively. Following 15 and 20 mg/kg p.o. dose, bioavailability was 102% and 117%, respectively. The harmonic mean of the corresponding terminal half-lives (T(1/2) λ(z) ) across all the dose groups was 1.71 h. The C(max) /MIC(90) and AUC(0∞24) /MIC(90) for the 15 and 20 mg/kg p.o. doses were ≥5.22 and ≥8.98, and ≥25.80 and ≥39.37 h, respectively. The results of this study suggest that 20 mg/kg orbifloxacin p.o. would be a rational daily dose to treat susceptible infections in Japanese quail not intended for food consumption. For more sensitive bacterial organisms, 15 mg/kg p.o. may also be effective.

Comparative adrenocortical suppression in dogs with otitis externa following topical otic administration of four different glucocorticoid-containing medications.

The safety of using otic formulations is often of concern for practitioners and pet owners alike, with "safe" in this context meaning no adrenocortical suppression. This study evaluated the effect of four glucocorticoid-containing otic formulations on plasma cortisol concentrations, measured by corticotropin stimulation testing (plasma cortisol concentrations before and after corticotropin injection), in dogs presented with otitis externa. Dexamethasone tended to have larger adrenocortical suppression compared with the other three formulations (betamethasone, triamcinolone, and mometasone), but the difference was not statistically significant. The largest difference among the four drugs was observed between dexamethasone and betamethasone (P=.09).

A phase II trial evaluating the safety and effectiveness of the AastromReplicell system for augmentation of low-dose blood stem cell transplantation.

To reduce the number of apheresis procedures and maintain the usual rate of hematopoietic recovery in patients treated with high-dose chemotherapy, we studied the effect of adding a small volume of ex vivo expanded bone marrow to low doses of CD34(+) blood stem cells. Thirty-four patients with breast cancer received G-CSF (10 microg/kg/day) priming followed by a limited volume (50-100 ml) bone marrow aspiration and standard 10-liter aphereses. Marrow was expanded ex vivo using the AastromReplicell system and infused along with low doses of blood-derived CD34(+) cells, collected in one apheresis. Thirty-one evaluable patients received a median CD34(+) blood stem cell dose of 0.7 x 10(6)/kg (range, 0.2-2.5) and 4.7 x 10(7) nucleated cells/kg (range, 1.98-8.7) of ex vivo expanded marrow. All patients recovered with normal blood counts and engrafted 500 neutrophils/microl and 20 000 platelets/microl in a median of 10 and 13 days, respectively. Multivariate analysis revealed that, in addition to CD34(+) lineage negative cell quantity, the quantity of stromal progenitors contained in the ex vivo expanded product correlated with engraftment outcome (r = 0.551, P = 0.004). Our results indicate that ex vivo expanded bone marrow is capable of facilitating engraftment when combined with low doses of mobilized blood derived CD34(+) cells.

Prompt and durable engraftment in two older adult patients with high risk chronic myelogenous leukemia (CML) using ex vivo expanded and unmanipulated unrelated umbilical cord blood.

Delayed engraftment, graft failure, and adverse transplant-related events have been observed in unrelated umbilical cord blood (UCB) recipients, particularly in those receiving a low leukocyte cell dose and in CML patients. We report the outcomes of two older adult patients with high risk CML who received a low leukocyte cell dose of unmanipulated UCB cells supplemented with ex vivo expanded (AastromReplicell System) UCB cells. Each engrafted promptly and neither patient experienced GVHD or life-threatening infection. Both remain engrafted with cells exclusively of donor origin and are in cytogenetic remission at 19 and 8 months follow-up. Ex vivo expanded UCB cells appear to facilitate hematopoietic recovery and therefore may increase the number of CML patients eligible for unrelated UCB transplant.

Autologous transplantation of ex vivo expanded bone marrow cells grown from small aliquots after high-dose chemotherapy for breast cancer.

The collection of small aliquots of bone marrow (BM), followed by ex vivo expansion for autologous transplantation may be less morbid, and more cost-effective, than typical BM or blood stem cell harvesting. Passive elimination of contaminating tumor cells during expansion could reduce reinoculation risks. Nineteen breast cancer patients underwent autotransplants exclusively using ex vivo expanded small aliquot BM cells (900-1200 x 10(6)). BM was expanded in media containing recombinant flt3 ligand, erythropoietin, and PIXY321, using stromal-based perfusion bioreactors for 12 days, and infused after high-dose chemotherapy. Correlations between cell dose and engraftment times were determined, and immunocytochemical tumor cell assays were performed before and after expansion. The median volume of BM expanded was 36.7 mL (range 15.8-87.0). Engraftment of neutrophils greater than 500/microL and platelets greater than 20,000/microL were 16 (13-24) and 24 (19-45) days, respectively; 1 patient had delayed platelet engraftment, even after infusion of back-up BM. Hematopoiesis is maintained at 24 months, despite posttransplant radiotherapy in 18 of the 19 patients. Transplanted CD34(+)/Lin(-) (lineage negative) cell dose correlated with neutrophil and platelet engraftment, with patients receiving greater than 2.0 x 10(5) CD34(+)/Lin(-) cells per kilogram, engrafting by day 28. Tumor cells were observed in 1 of the 19 patients before expansion, and in none of the 19 patients after expansion. It is feasible to perform autotransplants solely with BM cells grown ex vivo in perfusion bioreactors from a small aliquot. Engraftment times are similar to those of a typical 1000 to 1500 mL BM autotransplant. If verified, this procedure could reduce the risk of tumor cell reinoculation with autotransplants and may be valuable in settings in which small stem cell doses are available, eg, cord blood transplants. (Blood. 2000;95:2169-2174)

Electrochemical sensor for measurement of urea and creatinine in serum based on ac impedance measurement of enzyme-catalyzed polymer transformation.

Enzyme-catalyzed polymer transformation with electrochemical ac impedance detection has been employed for the measurement of urea and creatinine in serum samples. A polymer, based on poly(methylvinyl ether)/maleic anhydride modified by esterification with n-octanol, which is stable at pH 7.4 and which is transformed rapidly in response to alkaline pH changes, was linked to enzymatic reactions between urease and urea or creatinine deiminase and creatinine to produce a disposable sensor system. The polymer was screen-printed onto interdigitated screen-printed carbon electrodes and the electrodes overlaid with absorbent pads containing the relevant enzyme. Application of serum samples, "spiked" with either urea or creatinine, resulted in rapid polymer transformation, and resultant changes in the capacitance of the polymer-coated electrodes were analyte-concentration dependent. Additional information on the mechanisms of polymer transformation was obtained from dynamic quartz crystal microbalance measurements.

Clinical-scale human umbilical cord blood cell expansion in a novel automated perfusion culture system.

Use of umbilical cord blood (CB) for stem cell transplantation has a number of advantages, but a major disadvantage is the relatively low cell number available. Ex vivo cell expansion has been proposed to overcome this limitation, and this study therefore evaluated the use of perfusion culture systems for CB cell expansion. CB was cryopreserved using standard methods and the thawed unpurified cells were used to initiate small-scale cultures supplemented with PIXY321,flt-3 ligand, and erythropoietin in serum-containing medium. Twelve days of culture resulted in the optimal output from most CB samples. Frequent medium exchange led to significant increases in cell (93%), CFU-GM (82%) and LTC-IC (350%) output as compared with unfed cultures. As the inoculum density was increased from 7.5 x 10(4) per cm2 to 6.0 x 10(5) per cm2, the output of cells, CFU-GM, and LTC-IC increased. Cell and CFU-GM output reached a plateau at 6.0 x 10(5) per cm2, whereas LTC-IC output continued to increase up to 1.2 x 10(6) per cm2. Because the increase in culture output did not increase linearly with increasing inoculum density, expansion ratios were greatest at 1.5 x 10(5) per cm2 for cells (6.4-fold) and CFU-GM (192-fold). Despite the lack of adherent stroma, CB cultures expressed mRNA for many growth factors (G-CSF, GM-CSF, IL-1, IL-6, LIF, KL, FL, Tpo, TGF-beta, TNF-alpha, and MIP-1alpha) that were also found in bone marrow (BM) cultures, with the exception of IL-11 (found only in BM) and IL-3 (found in neither). Culture output was remarkably consistent from 10 CB samples (coefficient of variation 0.3) indicating that the procedure is robust and reproducible. Two commercial serum-free media were evaluated and found to support only approximately 25% of the culture output as compared with serum-containing medium. Implementation of optimal conditions in the clinical scale, automated cell production system (CPS) showed that the process scaled-up well, generating 1.7 x 10(7) CFU-GM (298-fold expansion) from 1.2 x 10(8) thawed viable nucleated CB cells (n = 3). The ability to generate >10(7) CFU-GM from <15 ml of CB within this closed, automated system without the need for extensive cell manipulations will enable clinical studies to test the safety and efficacy of expanded CB cells in the transplant setting.

A field evaluation of an indirect fluorescent antibody-based broodstock screening test used to control the vertical transmission of Renibacterium salmoninarium in Chinook salmon (Oncorhynchus tshawytscha).

Ovarian fluid samples from erythromycin treated and untreated spawning three year old Chinook salmon were screened independently by two laboratories for the presence of Renibacterium salmoninarum using the indirect fluorescent antibody technique (IFAT). Agreement between the results of the two laboratories could be explained by chance when R. salmoninarum cell numbers as low as one per sample were considered sufficient to represent a positive result. If a positive result was considered to be the detection of larger numbers of R. salmoninarum cells (greater than 51 cells per sample), agreement increased and there was a statistically significant association between the results of the two laboratories. However, the level of agreement did not reach satisfactory levels for a population screening test. Furthermore, approximately 60% of the samples yielded false negative results when IFAT results were compared with positive culture results. These results led to the conclusion that the IFAT screening procedure, as carried out, was unsuitable for the purposes intended. Erythromycin injection of the spawning fish had no statistically significant effect on the results of the IFAT screening test.

In vitro evaluation of 6-thioguanine and alpha-interferon as a therapeutic combination in HL-60 and natural killer cells.

The effect of 6-thioguanine (6-TG) and alpha-interferon (IFN-alpha) was evaluated in vitro to determine their effectiveness in combination on the therapeutically relevant events of: HL-60 cell cytotoxicity, HL-60 cell differentiation, and natural killer (NK)-cell mediated cytotoxicity. 6-TG was toxic to HL-60 cells (ID50 = 0.6 microM; 24-h exposure) while IFN-alpha (up to 1000 IU/ml) had minimal cytotoxic activity. Sequence-dependent activity was observed, inasmuch as the IFN-alpha pretreatment sequence was antagonistic, while the other schedules were additive or, possibly, synergistic. The combination of 0.5 microM 6-TG and 100 IU/ml IFN-alpha produced the same level of HL-60 cell differentiation as each agent alone, suggesting no benefit from the combination on this process. The effect of 6-TG and IFN-alpha on NK cell-mediated cytotoxicity was found to be sequence dependent. NK cell activity was markedly stimulated by IFN-alpha, whereas 6-TG alone seemed to have no direct effect. However, when the NK cells were pretreated with 100 IU/ml IFN-alpha followed by 10 microM 6-TG, the IFN-alpha-enhanced activity of NK cells was ablated. These results suggest that the immunosuppressive activity of 6-TG may be related to the acute inhibition of cytokine activation. Our results suggest that 6-TG and IFN-alpha have considerable interactions, which are sequence dependent. The optimal sequence for potential therapeutic application of these anticancer agents appears to be 6-TG pretreatment followed by IFN-alpha.

Modulation of 6-thioguanine activity by guanine in human promyelocytic leukemia HL-60 cells.

The effects of guanine coadministration on the metabolism and biological activity of 6-thioguanine (6-TG) were studied in human promyelocytic leukemia cells (HL-60). Cell growth, cytotoxicity (cloning assay), and cell differentiation were measured, along with nucleotide metabolism. Guanine was efficiently salvaged by HL-60 cells; at 200 microM, guanine suppressed the formation of 6-TG mononucleotides and abolished 6-TG incorporation into nucleic acids. Similarly, guanine antagonized 6-TG cytotoxicity in a dose dependent fashion. Furthermore, guanine (200 microM) fully suppressed the 6-TG (10 microM) induced HL-60 cell differentiation, which suggests that cell differentiation at pharmacological 6-TG concentrations is dependent on the anabolism of the drug to active nucleotides. 6-TG given alone reduced GTP levels and DNA synthesis rates in HL-60 cells, while a major intracellular 6-TG metabolite, 6-thioguanosine 5'-monophosphate, accumulated to high levels (approximately 100 microM). It is suggested that accumulation of 6-thioguanosine 5'-monophosphate and a resultant partial block of the de novo biosynthesis of guanine nucleotides is responsible for 6-TG induced cell differentiation in HL-60 cells.