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Conclusions: Topical administration of fluoroquinolonecontaining solutions results in measurable differences in laser-induced corneal fluorescence. Under these experimental conditions, 0.3% ofloxacin, 0.5% levofloxacin, and 0.3% gatifloxacin all appear to penetrate the epithelium significantly more than 0.3% ciprofloxacin P .02 ; . Clinical Relevance: Monitoring of laser-induced fluorescence may be helpful in determining the penetration depths and concentrations of topically applied fluoroquinolones within the cornea.
Pain varies from person to person. When it is severe, it can limit your daily routines at home and at work. Also, it can get in the way of how you feel in general about your well-being. Steriod injections: Steroids corticosteroids ; can be injected by a needle into and around joints to relieve pain and swelling. Many different types of steroids can be injected. Steroids can also be injected into different parts of the joint or around the joint into muscles and tendons. Where the injection is put depends on where the doctor thinks the problem is.
The SF-36 comprises 36 items that record general well-being during the previous 30 d 22 ; The items are formulated as statements or questions and were scored as numbers. Eight parameters were calculated with a range of 0 100: physical problems, bodily pain, general health, vitality, social functioning, emotional role, and mental health. The first three parameters measure physical health, the last three parameters measure mental health, and the general health and vitality scales are sensitive to both physical and mental health outcomes. Higher scores represent better quality of life 23.
References 1. Davey R: Of gestational diabetes, finesse, and an antipodean snark Letter ; . Diabetes Care 22: 873874, 1999 Moses R, Griffiths R, McPherson S: The incidence of gestational diabetes in the Illawarra area of New South Wales. Aust N Z J Obstet Gynaecol 34: 425427, 1994 Beischer N, Oats JN, Henry OA, Sheedy MT, Walstab JE: Incidence and severity of gestational diabetes according to country of birth in women living in Australia. Diabetes 40 Suppl. 2 ; : 3538, 1991 4. Moses R, Moses M, Russell K, Schier G: The 75-g glucose tolerance test in pregnancy: a reference range determined on a low-risk population and related to selected pregnancy outcomes. Diabetes Care 21: 18071811, 1998 Moses RG: Is it time to modify the glucose tolerance test for the diagnosis of gestational diabetes? Letter ; Diabetes Care 18: 886, 1995 Moses RG, Schier GM: Glucose tolerance testing and gestational diabetes Letter ; . Med J Aust 166: 108, 1997.
Macrolides can influence several neutrophil cell functions. In numerous in vitro and ex vivo studies, it was shown that macrolides inhibit oxidant production.32 37 In addition, macrolides promote neutrophil cell degranulation in vitro, 38, 39 and ex vivo.40 Furthermore, macrolides reduce neutrophil cell phagocytosis ex vivo.37 Additionally, macrolides increase neutrophil cell migration in healthy volunteers, 41 and in patients with persistently abnormal neutrophil cell chemotaxis.42 Macrolides have been found to have anti-inflammatory effects in vivo in several animal models. A series of animal investigations have studied the effects of macrolides on pulmonary host defence: in one study, the effect of erythromycin on intrapulmonary accumulation of neutrophil cells after intra-tracheal challenge with interleukin IL ; -8 or lipopolysaccharide LPS ; was studied in mice.15 Intrapulmonary neutrophil cell accumulation was significantly suppressed after intraperitoneal adminis.
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P133 TheNovelTransgenicMouse, C3B6-Trp53tm1Brd[N12]F1, Permits 45-wk Cancer Screening for Transplacental and Neonatal Exposure to Zidovudine SM Lewis * 1, FW Lee1, JK Dunnick2, WT Allaben1, C Weis1, JE Leakey1 of Scientific Coordination, National Center for Toxicological Research, Jefferson, AR; 2 NTP, NIEHS, Research Triangle Park, NC The nucleoside reverse transcriptase inhibitor NRTI ; drug zidovudine AZT ; is given to HIV-infected women during pregnancy and to neonates to reduce maternal-fetal HIV transmission. AZT is carcinogenic in mice and genotoxic in fetal mice. To examine toxicity during transplacental and neonatal exposure, the C3B6-Trp53tm1Brd[N12]F1 transgenic mouse model mimics human in utero and oral dosing. The mouse, haplodeficient for the p53 tumor suppressor gene, develops tumors at an increased rate due to partial p53 gene loss. It has demonstrated robust reproductive and survival traits. Mice, n 27 per group, were gavaged daily with 0 vehicle control ; , 80, 160, or 240 mg kg AZT in aqueous methylcellulose Tween 80 0.2 0.1% ; transplacentally from gestation day 12 to 18, and from postnatal day PND ; 1 to 30 age. Vehicle control and 240 mg kg AZT survival at PND28 were 98.7% and 96.5%, respectively. At 45 wk age, control males and females averaged 88.9% overall survival; 240 mg kg AZT male survival was 81.5% as compared to female survival 77.7% ; . Pathology findings at 30 wk suggest dose sensitivity to carcinogenic effects with 23% of tumors, predominantly lymphomas, observed in 240 mg kg AZT-dosed males, but no significant female effects. Preliminary results also suggest that in 45-wkold mice, greater numbers of tumors were observed with increasing dose; liver tumors were present in the higher doses. This C3B6-Trp53tm1Brd[N12]F1 model allows for rapid toxicity and carcinogenic screening of NRTIs and permits chronic bioassays to be completed within a 12-mo period as compared to standard 2-y bioassays.
In a dose-dependentmanner with single-hit kinetics. Liquidsuspension preincubationof blood cells with FK506 before culture in methylcellulose induced a significant increase in the amount of IL-3-supported growth of CFU-GM and BFUE, whereas initial preincubation with IL-3 and subsequent culture with FK506 plus IL-3 exerted its stimulatory effect only on BFU-E. These data suggest that the stimulation of hematopoietic progenitor cells by FK506 occurs at a very early stage of maturation and diminishes with further myeloid development. 0 1993 by The American Society of Hematology and methysergide.
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Were separated from PB or CB samples by density-gradient centrifugation using lymphocyte separation medium Organon Teknika, Durham, NC ; after phagocytes were depleted with silica Immuno Biological Laboratories, Fujioka, Japan ; . The interface containing mononuclear cells was collected after density-gradient centrifugation. Lineage-negative Lin ; cells were negatively selected from the mononuclear cells using a magnetic separation column MACS II; Miltenyi Biotec, Bergisch Gladbach, Germany ; and a mixture of magnetic microbead-conjugated antibodies against CD4, CD8, CD11b, CD14, and CD19 Miltenyi Biotec ; according to the manufacturer's instructions. In some experiments, CD34 cells were positively selected from CB or bone marrow mononuclear cells using the CD34 cell isolation kit Miltenyi Biotec ; . Cytokines and antibodies The following cytokines were added to the cell suspension. Recombinant IL-3 was purchased from Intergen Purchase, NY ; , and rIL-6 was kindly provided by Kirin Brewery Maebashi, Japan ; . Bulk vials of rSCF were purchased from PeproTech EC London, United Kingdom ; , and recombinant IL-4 was purchased from R&D Systems Minneapolis, MN ; . Cell culture Cells were suspended in Iscove modified Dulbecco medium IMDM; Gibco BRL, Grand Island, NY ; supplemented with 1% insulintransferrin selenium Gibco BRL ; , 50 M 2-ME Gibco BRL ; , 1% penicillin streptomycin Gibco BRL ; , and 0.1% bovine serum albumin complete IMDM; Sigma, St Louis, MO ; . For methylcellulose culture, the Lin 106 PB or 105 CB mononuclear cells or 103 CB or bone marrow CD34 cells were suspended in 0.3 mL complete IMDM. The cells were mixed by shaking the tubes for more than 1 minute with 2.7 mL serum-free Iscove methylcellulose medium MethoCult SFBIT; Stem Cell Technologies, Vancouver, Canada ; supplemented with 200 ng mL SCF, 50 ng mL IL-6, and 1 ng mL IL-3. IL-3 was added only at the beginning of culture because it efficiently enhances MC colony formation without inducing the other cell type colonies as has been reported.9, 13 The cell suspension was inoculated at 0.3 mL per well in the 24-well plate Iwaki Glass, Tokyo, Japan ; at 37C in 5% CO2. Every 2 weeks, 0.3 mL fresh methylcellulose medium containing 100 ng mL SCF and 50 ng mL IL-6 was layered over the methylcellulose cultures. At 6 weeks, whole cells were retrieved after methylcellulose medium was dissolved with PBS. They were then suspended and cultured in complete IMDM supplemented with 100 ng mL SCF, 50 ng mL IL-6, and 5% fetal calf serum Cansera, Rexdale, Canada ; in 25-cm2 flasks Iwaki Glass ; up to the 25th week. Although MCs cultured initially in methylcellulose were used in most of the experiments, we used some CB-derived MCs cultured initially in the cytokine-supplemented IMDM liquid medium in large-scale experiments cytokine assay and GeneChip [Affymetrix, Santa Clara, CA] analysis ; by mixing them with MCs cultured initially in methylcellulose. We confirmed each time that these MCs did not largely differ in terms of Fc RI expression. Staining The differential count of cultured cells was determined based on 100 cells, unless smears had fewer cells, by using cultured samples centrifuged onto slides with Cytospin 2 Shandon, Pittsburgh, PA ; . Cells were examined with May-Grunwald and Giemsa staining or with anti-tryptase immunostain ing. Immunostaining for human mast cell tryptase was performed by using the method previously described.9-12 Briefly, the smears were fixed with Carnoy solution 60% ethanol, 30% chloroform, 0% glacial acetic acid ; and stained for granular tryptase by the alkaline phosphatase anti-alkaline phosphatase APAAP ; method with the DAKO Carpinteria, CA ; APAAP kit according to the manufacturer's instructions. Histamine release assay MCs were sensitized with 1 g mL human myeloma IgE a generous gift from Dr Kimishige Ishizaka, La Jolla, CA ; at 37C for 48 hours in the absence or presence of IL-4. After they were washed, cells were suspended at a density of 105 cells mL in modified Tyrode solution pH 7.4 ; containing and metolazone.
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Tations or deletions could occur at various sites in the cellular DNA. Furthermore, the AID gene, which is used by normal B cells to create Ig V gene mutations and to delete DNA segments during isotype switching, can be expressed in B-CLL cells.27-29 Because AID is expressed intermittently in only a small fraction of the B-CLL clone, 29 new genetic defects developing through the action of this enzyme would be diverse in nature and would exist only among certain clonal members. However, it should be clear that at this juncture there are no firm data to implicate AID gene expression with ongoing SHM or genome instability. Clinical Implications of These Findings Some patients with B-CLL survive for decades after initial diagnosis and never require therapy; others succumb rapidly to the disease despite therapy. To deal with this heterogeneity in clinical course and outcome, Rai et al56 and subsequently Binet et al57 created staging systems based on clinical symptoms, physical signs, and laboratory values. These systems currently represent the gold standards for patient evaluation and treatment decisions. Some of the findings mentioned above have led to the recognition of subgroups of patients, at the laboratory level, that are proving to be valuable adjuncts to the Rai and Binet staging systems. Ig V gene mutation status is currently the most accurate discriminator of clinical outcome in several retrospective studies.14-16, 54, 58-60 Patients with few mutations succumb to the disease at least 3 times faster than those with significant mutations approximate median survivals 8 years versus 24 years ; . Two protein markers that are easier to measure in the clinical laboratory that have prognostic value are intracellular ZAP-7044, 61, 62 and cell surface CD38.16, 59, 63 An inverse correlation exists between each of these markers and clinical course and survival. Since CD38 levels may change over time in some patients15, 59, 63, 64 and may correlate with increased disease aggressiveness, 64 this marker may also be helpful in determining a worsening of clinical course. At the present time, it appears that ZAP-70 correlates well with Ig V gene mutations and therefore may be a convenient clinical surrogate for V gene mutation status.61 B-CLL cells in both the high-risk Ig V gene subgroup and the CD38 subgroup13-15, 52, 54 are more likely to express ominous cytogenetic abnormalities e.g., trisomy 12, 11q-, and 17p- ; . Level of Leukemia Cell Turnover In Vivo The rate at which any biological system evolves is determined by the frequency that variants are produced and the relative selective advantages that the variants possess. For newly arisen subclones to become a sigAmerican Society of Hematology.
Retroviral transduction and transplantation assays Hematopoietic progenitor transformation assays were performed as previously described.15 Briefly, viral supernatants were collected 3 days after transfection of Phoenix cells and used to infect hematopoietic stem and progenitors cells harvested from the bone marrow of 4- to 10-week-old C57BL 6 mice ; that were positively selected for c-Kit expression by magnetic-activated cell sorting MACS ; . After spinoculation, transduced cells were then plated in 1% methylcellulose Stem Cell Technologies, Vancouver, BC, Canada ; supplemented with cytokines in the presence or absence of 1 mg mL G418. After 7 days of culture, colonies were counted to calculate the transduction efficiency. Single-cell suspensions 104 cells ; of G418-resistant colonies were then replated in methylcellulose medium supplemented with growth factors without G418. Plating was repeated every 7 days. In each round of replating, single-cell suspensions were also expanded in RPMI liquid culture containing 20% fetal calf serum FCS ; plus 20% WEHI-conditioned medium. For tumorigenicity assays, 106 immortalized cells were injected into the retro-orbital venous sinus of 6-week-old syngeneic C57BL 6 mice, which had received a sublethal dose of 5.25 Gy total body irradiation 135Cs ; . Mice were maintained on antibiotic water to avoid infection and monitored for development of leukemia by complete blood count, blood smear, and fluorescenceactivated cell sorting FACS ; analysis. Tissues were fixed in buffered formalin, sectioned, and stained with hematoxylin and eosin for histologic analysis. Phenotype analysis Immunophenotypic analysis was performed by FACS using fluorochrome-conjugated monoclonal antibodies to Sca-1 D7 clone ; , c-Kit 2B8 clone ; , Mac-1 M1 70 clone ; , Gr-1 RB6-8C5 clone ; , B220 RA3-6B2 clone ; , and CD19 1D3 clone ; , all from Pharmingen San Diego, CA ; , respectively. Staining was generally performed on ice for 15 minutes. Cells were washed twice in staining medium and resuspended in 1 g propidium iodine PI ; before analysis using a Moflops a modified triple laser Cytomation Becton Dickinson hybrid FACS ; . Dead cells were gated out by high PI staining and forward light scatter. Expression studies Reverse transcriptionpolymerase chain reaction RT-PCR ; was performed using MLL and fusion genespecific primers sequences available on request ; on total RNA extracted from primary transduced bone marrow cells. Western blotting was performed on COS7 or Phoenix cells transiently transfected with various MLL constructs. Lysate proteins 30 g ; were fractionated in 5% polyacrylamide gel and transferred to enhanced chemiluminescence ECL ; membranes Amersham Pharmacia Biotech, Piscataway, NJ ; using Tris tris hydroxymethyl ; aminomethane ; glycine sodium dodecyl sulfate SDS ; transfer buffer. After blocking, membranes were probed with monoclonal antibody N4.4 directed against an MLL aminoterminal epitope as previously described.14, 15 Transcriptional transactivation assays The 293 or COS7 cells 5 104 ; were seeded overnight in 24-well plates before transfection using Fugene 6 Roche Molecular Biochemicals, Indianapolis, IN ; . Then, 0.2 g Gal4-DBD fusion construct was cotransfected with 0.1 g pcDNA3.1 LacZ internal control plasmid and 0.2 g luciferase reporter construct, which contained 2 tandem copies of Gal4 consensus-binding sites and the luciferase gene driven by herpes simplex virus thymidine kinase HSV-TK ; , adenovirus E1b, or the myelomonocytic growth factor promoters.17 Luciferase activities were normalized based on -galactosidase levels. Means and SDs were determined from at least 3 independent experiments performed in duplicate and micafungin.
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L. W. Dow et al. Cells and Culture Conditions. The growth characteristics of wild-type CEM cells have been described 13 ; . All cells were grown in Eagle's medium modified for suspension cultures and supplemented with 10% heat-inactivated fetal calf serum. CEM cells were selected for resistance to ara-C by growth in the continuous presence of sublethal concentrations of the drug. ara-C is highly toxic to CEM cells, a concentration of 2 x 10~8 M being sufficient to reduce cloning efficiency by 50%. After about 3 months of repeated culturing of surviving cells in increasing concentrations of ara-C, a subclone designed CEM ara-C was isolated in 0.8% methylcellulose Methocel; Dow Chemical Co., Midland, Mich. ; . Single colonies were removed with a micropipet and with the aid of inverted microscopy and were grown in microtitration plates Linbro Scientific Inc., Hamden, Conn. ; until sufficient cells were obtained for transfer to flasks. Since about 4 x 10~5 M ara-C is necessary to reduce the plating efficiency of CEM ara-C by 50%, this subclone is about 2000 times more resistant to ara-C than are the wildtype cells. This increased resistance to ara-C correlated with a marked deficiency of deoxycytidine kinase. The level of deoxycytidine kinase in drug-resistant cells was less than 1% of the activity 7.1 nmol dCMP per hr per mg protein ; present in wild-type CEM cells. Cytotoxicity Assays. Clonal assays were used to determine cytotoxicity 28, 30 ; . Cells, 3 x 105 ml, were incubated in F14 medium plus 10% heat-inactivated fetal bovine serum KC Biological, Tenexa, Kans. ; in the presence or absence of Fara-A or ara-A with deoxycoformycin 0.5 ig ml ; for 18 hr at CO2 atmosphere. Cells were washed twice and were then resuspended in semisolid medium. Triplicate 1-ml cultures of 103 cells ml in 0.8% methylcellulose 32 ; , 10% fetal bovine serum, and 4 x 10~5 M dCyd were incubated in 35-mm Retri dishes Lux; Flow Laboratories, Rockville, Md. ; in a humidified 5% CO2 atmosphere. Colonies reached maximal size in 11 days, and those containing more than 50 cells were counted with the aid of inverted microscopy. Cloning efficien cies ranged from 24 to 94%, and standard deviations were 10% or less. The results are expressed as percentages of colony formation by control cultures. Cell Preparation and Flow Cytometric Analysis. After in cubation in drug, the cell samples were divided into 2 aliquots, one for cytometric analysis and the other for survival studies. Cells for cytometric analysis were centrifuged at 1000 rpm for 5 min at room temperature; the medium was then discarded and the cell pellet was resuspended and stained with 1 ml of hypotonie propidium iodide citrate solution with gentle pipetting 20 ; . The nuclear suspension was incubated on ice for a minimum of 15 min before being analyzed. Immediately before analysis, the cell samples were vortexed and passed through a 37- im nylon mesh filter Small Parts, Inc., Miami, Fla. ; . Distributions of DMA content were determined with a Coulter TPS-1TC cell sorter. A 488-nm argon ion laser line was used to excite fluorescence, and the total fluorescence 590 nm ; from each cell was quantified and displayed on a 128-channel pulse-height analyzer. From 50, 000 to 100, 000 cells sample were analyzed for the DMA distribution analyses, regardless of the number of dead cells present in the sample. Fluorescent latex beads Coulter Electronics, Inc., Hialeah, Fla. ; were used to standardize the instrument before cell samples were ana lyzed. The coefficient of variation for the latex beads ranged from 4.5 to 5.5%, while that of the G, peak of control CEM 1406 cells ranged from 4.6 to 7.1%. Incorporation of [mef iy -3H]dThd into DNA. Samples 2 ml.
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Tion only occurred when cells were transfected with XbaI and HindIII digests of pCM4000, whereas EcoRI digests of the plasmid could not transform. Transformation of NIH 3T3 cells by pCM4000 did not occur at any greater frequency than with pCM1058, pCM1, or pJN201. These results precisely locate the transforming region between 0.123 and 0.14 map units on the AD169 genome. Characteristics of the transformed cells. NIH cells transformed by XbaI and HindIII digests of AD169 and pCM1058 DNA were picked from methylcellulose and grown in sufficient quantities for further studies. The cell lines established were sensitive to trypsin and maintained a fibroblastic morphology. Two cell lines, XM8 and 1058-3, were tested for replating efficiency in methylcellulose and formed colonies with efficiencies of 52 and 30%o, respectively. These efficiencies of replating were much higher than were the efficiencies found by Krontiris and Cooper 31 ; with NIH 3T3 cells transformed by bladder carcinoma DNA but lower than cells transformed by HSV-2 DNA 16 ; . To determine whether the cells transformed by AD169 and pCM1058 DNA were oncogenic, approximately 5 x 106 cells were inoculated intraperitoneally and subcutaneously into 3week-old athymic BALB c nude mice. The results of these experiments are summarized in Table 4. Twelve cell lines isolated at various passage levels were injected into nude mice. All of the transformed cell lines except one were tumorigenic in 100% of the inoculated mice. Palpable tumors were observed between 2 and 3.
Resulted swollen hydrogel is aqueous based, it is very comfortable in the human eye. Among these polymers, in situ gels are preferred since they are conveniently dropped in the eye as a solution, where undergo transition into a gel. Thermosensitive, specific ion sensitive or pH- sensitive hydrogels have been examined for their potential as vehicles for ocular drugs. Poloxamers as thermogelling polymers could be applicable for the development of effective ophthalmic drug delivery 30 ; . In order to reduce the concentration of polymer and or to achieve a phase transition temperature higher than room temperature 25C ; and gelling at precorneal temperature 35C ; , the combining Pluronic analogs 30 ; or the addition of further polymer, e. g. PEG 31 ; , PAA 32 ; , methylcellulose MC ; , HPMC, CMC 33 ; is often necessary. An alternative in situ gelling material of natural origin, xyloglucan, was evaluated for the sustained ocular delivery of pilocarpine 34 ; and timolol 35 ; . Ion-sensitive polymers belong to the mainly used in situ gelling materials for ocular drug delivery. Slightly viscous gellan gum solutions in low concentrations 1% ; show markedly increase in apparent viscosity, when introduced into presence of a physiological level of cations, without requiring more ions than 1025% of those in tear fluid 36 ; . The precorneal contact times for drugs can thus be extended up to 20-h 37 ; . Gellan containing formulations of pilocarpine HCl allowed reduction of drug concentration from 2% to 0.5% obtaining the same bioavailability 38 ; . The ability of gel formation at physiological Ca2 + levels was used in case of alginic acid as well. Presence of this polymer significantly extended the duration of the pressure reducing effect of pilocarpine to 10-h 39 ; and carteolol to 8-h 40 ; allowing only once a day administration in case of carteolol. Aqueous solutions of PAA that transform into gels upon increase in pH may be used as in situ gelling ophthalmic drug delivery systems. However, the amount of PAA required to form stiff gel upon instillation in the eye is not easily neutralized by the buffering action of tear fluid. Combination PAA with a suitable viscosity enhancing polymer e. g. HPMC 41 ; or MC allows a reduction in the PAA concentration without comprising the in situ gelling properties. The formulation containing Carbopol 940 and Methocel E50LV HPMC ; afforded sustained release of ofloxacin over an 8-h period 41 ; . Peroral drug delivery The pH-sensitive hydrogels have a potential use in site-specific delivery of drugs to specific regions of and mifeprex.
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