An in vitro study of the susceptibilities and growth dynamics of common ocular pathogens using five fluoroquinolones

Bacteria are responsible for up to 70% of all ocular infections including conjunctivitis, keratitis and endophthalmitis. If left untreated, a reduction of visual acuity, and in severe cases, sight loss, is possible. Treatment usually consists of a topically applied antibacterial preparation for patients with superficial infections. With intraocular infections, topical administration is augmented with systemic treatment or local instillation. While several types of drugs are available for ocular therapy, the fluoroquinolone class of antimicrobials is especially effective. This is due in part to their broad-spectrum of activity and low toxicity. However, as with any globally prescribed antimicrobial agent, bacterial resistance is an issue. Over the past 10 years there has been a decline in the effectiveness of older fluoroquinolones (ciprofloxacin and ofloxacin) in treating Gram-positive and, to a lesser extent, certain Gram-negative infections. In response to the declining activity of ciprofloxacin and ofloxacin, newer fluoroquinolones have been developed such as levofloxacin (L-isomer of ofloxacin), and more recently, gatifloxacin and moxifloxacin. In order to ensure the most potent drugs are being used to treat the most serious types of infection, studies need to be done to assess the activity of the current antimicrobial arsenal against pertinent infecting organisms. Three different types of experiments can be done to achieve this. In vitro potency can be tested two ways. The first is minimum inhibitory concentration (MIC). This test defines the concentration of antimicrobial drug that prevents growth of bacteria when tested against an inoculum of approximately 105 colony forming units (CFU)/ml. The second is the mutant prevention concentration (MPC), which is the amount of drug needed to inhibit a first step resistant mutant. This is a relatively new approach to measuring fluoroquinolone potency; like MIC it is not a measure of kill. A separate set of experiments are needed to assess in vitro killing. Kill curves measure the ability of an antimicrobial agent to reduce/kill a bacterial population over a period of 24 hours.Because bacterial loads can vary greatly in in vivo infections, kill curves were conducted on a series of four inoculum sizes ranging from 106 to 109 cfu/ml. Some of the most common ocular pathogens are Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae and Pseudomonas aeruginosa. Mycobacterium fortuitum and Mycobacterium chelonae, while much less commonly associated with ocular disease, are capable of causing vision-threatening infections. As a result, the above six organisms were used to test the in vitro potency of ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin and gatifloxacin.Both MIC and MPC testing found both gatifloxacin and moxifloxacin to be 4-8-fold more potent in vitro against the Gram-positive organisms than the older fluoroquinolones with an average potency rank order of moxifloxacin = gatifloxacin > levofloxacin > ofloxacin = ciprofloxacin. The Gram-negative results, however, revealed that the older fluoroquinolones are still the most potent of the fluoroquinolones tested with an average potency rank order of ciprofloxacin > ofloxacin = levofloxacin > gatifloxacin = moxifloxacin. Kill curve results showed a significant difference in the rate of killing between the MIC and MPC drug concentrations. At the MIC drug concentration there was generally only a noticeable reduction in viable cells following 24 hours of drug exposure and in many cases this was followed by a period of bacterial re-growth. At the MPC drug concentration, a significant bacterial count reduction was often observed as early as 4 to 6 hours for both S. pneumoniae and H. influenzae. Surprisingly, there was little difference between the five fluoroquinolones in their rates of and amount of bacterial reduction.Because of high in vitro resistance rates in drugs like penicillin, the fluoroquinolones are an important broad-spectrum alternative. Consequently, it is imperative that measures are taken to maintain the efficacy of this class. One approach is to ensure that the most potent drug is being used to eradicate possible resistant sub-populations present in in vivo infections. The data from these experiments suggest that the new fluoroquinolones gatifloxacin and moxifloxacin are much more potent (in vitro) than older fluoroquinolones against Gram-positive bacteria. With Gram-negative pathogens, however, ciprofloxacin remains the most potent agent in vitro

Analysis of health related quality of life (HRQoL) of patients with clinically localized prostate cancer, one year after treatment with external beam radiotherapy (EBRT) alone versus EBRT and high dose rate brachytherapy (HDRBT)

<p>Abstract</p> <p>Purpose</p> <p>Prostate cancer is the leading form of cancer diagnosed among North American men. Most patients present with localized disease, which can be effectively treated with a variety of different modalities. These are associated with widely different acute and late effects, which can be both physical and psychological in nature. HRQoL concerns are therefore important for these patients for selecting between the different treatment options.</p> <p>Materials and methods</p> <p>One year after receiving radiotherapy for localised prostate cancer 117 patients with localized prostate cancer were invited to participate in a quality of life (QoL) self reported survey. 111 patients consented and participated in the survey, one year after completion of their treatment. 88 patients received EBRT and 23 received EBRT and HDRBT. QoL was compared in the two groups by using a modified version of Functional Assessment of Cancer Therapy-Prostate (FACT-P) survey instrument.</p> <p>Results</p> <p>One year after completion of treatment, there was no significant difference in overall QoL scores between the two groups of patients. For each component of the modified FACT-P survey, i.e. physical, social/family, emotional, and functional well-being; there were no statistically significant differences in the mean scores between the two groups.</p> <p>Conclusion</p> <p>In prostate cancer patients treated with EBRT alone versus combined EBRT and HDRBT, there was no significant difference in the QoL scores at one year post-treatment.</p

Use of Non-Steroidal Anti-Inflammatory Drugs and Prostate Cancer Risk: A Population-Based Nested Case-Control Study

BACKGROUND: Despite strong laboratory evidence that non-steroidal anti-inflammatory drugs (NSAIDs) could prevent prostate cancer, epidemiological studies have so far reported conflicting results. Most studies were limited by lack of information on dosage and duration of use of the different classes of NSAIDs. METHODS: We conducted a nested case-control study using data from Saskatchewan Prescription Drug Plan (SPDP) and Cancer Registry to examine the effects of dose and duration of use of five classes of NSAIDs on prostate cancer risk. Cases (N = 9,007) were men aged ≥40 years diagnosed with prostatic carcinoma between 1985 and 2000, and were matched to four controls on age and duration of SPDP membership. Detailed histories of exposure to prescription NSAIDs and other drugs were obtained from the SPDP. RESULTS: Any use of propionates (e.g., ibuprofen, naproxen) was associated with a modest reduction in prostate cancer risk (Odds ratio = 0.90; 95%CI 0.84-0.95), whereas use of other NSAIDs was not. In particular, we did not observe the hypothesized inverse association with aspirin use (1.01; 0.95-1.07). There was no clear evidence of dose-response or duration-response relationships for any of the examined NSAID classes. CONCLUSIONS: Our findings suggest modest benefits of at least some NSAIDs in reducing prostate cancer risk

Planning target volume margins for prostate radiotherapy using daily electronic portal imaging and implanted fiducial markers

<p>Abstract</p> <p>Background</p> <p>Fiducial markers and daily electronic portal imaging (EPI) can reduce the risk of geographic miss in prostate cancer radiotherapy. The purpose of this study was to estimate CTV to PTV margin requirements, without and with the use of this image guidance strategy.</p> <p>Methods</p> <p>46 patients underwent placement of 3 radio-opaque fiducial markers prior to prostate RT. Daily pre-treatment EPIs were taken, and isocenter placement errors were corrected if they were ≥ 3 mm along the left-right or superior-inferior axes, and/or ≥ 2 mm along the anterior-posterior axis. During-treatment EPIs were then obtained to estimate intra-fraction motion.</p> <p>Results</p> <p>Without image guidance, margins of 0.57 cm, 0.79 cm and 0.77 cm, along the left-right, superior-inferior and anterior-posterior axes respectively, are required to give 95% probability of complete CTV coverage each day. With the above image guidance strategy, these margins can be reduced to 0.36 cm, 0.37 cm and 0.37 cm respectively. Correction of all isocenter placement errors, regardless of size, would permit minimal additional reduction in margins.</p> <p>Conclusions</p> <p>Image guidance, using implanted fiducial markers and daily EPI, permits the use of narrower PTV margins without compromising coverage of the target, in the radiotherapy of prostate cancer.</p

Invitro alteration of rat pancreatic islet immunogenicity in an allogeneic transplant model

Allograft rejection remains the fundamental stumbling block to tissue transplantation. Traditional assumption has been that transplanted tissue alone provides an antigen source (alloantigen), which directly stimulates a host response resulting in graft rejection; accordingly, traditional attempts at circumventing the allograft response have focussed on techniques of recipient immunosuppression. Recently, increasing attention has been given to a subset of non-parenchymal, bone marrow derived lymphoid cells (characterized by their surface expression of class II MHC antigen) which are carried passively with the allograft into an immune competent recipient. A current hypothesis is that these cells, called antigen presenting cells (APCs), participate in the sensitization of the immunologically naive but responsive host to the transplanted tissue, leading ultimately to graft rejection. Therefore, it has been suggested that depletion of APCs from donor tissue prior to transplantation may permit allogeneic transplantation to occur, without host immunosuppression. In contrast to solid organs, pancreatic islets are well suited to this type of immunomodulation prior to transplantation, since they can be maintained in a functional ex vivo state by cell culture. The purpose of this thesis was to evaluate donor islet APC depletion by pre-transplant cell culture and APC-ablative photodynamic therapy (PDT), and to see whether either in vitro technique could prevent rejection in a rat, allogeneic transplant model. Briefly, a donor (Sprague Dawley, RTlu) -recipient (Wistar Furth, RTla) pair with a major histo-incompatible barrier was selected. After collagenase digestion of donor pancreata, islets were isolated from A the digested tissue by centrifugation through a discontinuous dextran gradient followed by hand picking using a dissecting microscope. Once isolated, the islets were either used fresh, placed in tissue culture (Ham's F-12 media, 11 mM glucose, 5% C02/room air at 37 C) for variable periods, or subjected to APC-ablative PDT. Islet APC depletion was assessed by fluorescent immunocytochemistry. Fresh, cultured and PDT treated islets were frozen in liquid N2 then cryostat sectioned and stained for class II MHC + cells (APCs), using an anti-class II mouse monoclonal antibody (OX-6), followed by a fluorescent (fluorescein indothiocyanate) labelled anti-mouse monoclonal. Using this technique, APCs could be identified by fluorescent microscopy on the basis of their enhanced surface staining. While fresh islets demonstrated between 1 and 5 APCs per cryostat section, a culture period of at least 10 days resulted in complete islet APC elimination. Islet allograft studies with fresh and cultured islets were then performed to determine: 1) if pre-transplant islet culture could sufficiently reduce donor tissue immunogenicity to allow successful allografting in immune-competent recipients, and if so, 2) what duration of culture was necessary to permit consistently successful allografting. Allografts of fresh and cultured (4, 7, 10, 14, and 21 day) islets were placed under the renal capsule of immune-competent, recipient rats and after 12 days the grafts were removed and studied histologically for evidence of rejection. While all grafts which were cultured for 10 days or less prior to transplantation were rejected, 4/10-14 day cultured islets, and 4/5-21 day cultured islets demonstrated engraftment. In vivo function of 21 day cultured islet allografts was demonstrated by transplantation of islets via the portal vein, into recipients which had been rendered hyperglycemic by IV streptozotocin. This resulted in an immediate and sustained reversion to euglycemia (as assessed by daily plasma glucose determinations using a glucose analyser) over a 30 day period of study. In contrast, streptozotocin "diabetic" recipients of fresh and 14 day cultured islet allografts demonstrated a brief (7-10 day) period of graft function (euglycemia) prior to a return of hyperglycemia, consistent with graft rejection. Photodynamic therapy (PDT) achieves selective cell ablation by the stimulated emission of singlet oxygen from a light-activated compound (benzoporphyrin) which has been delivered to the cell target. In these experiments, APC elimination was attempted by in vitro islet treatment with OX-6, followed by a specific, secondary antibody (RAMIg) to which BPD had been conjugated. After UV lightactivation the treated islets were frozen, cryostat sectioned and immunostained for Class II MHC + cells. In contrast to control islets which underwent a secondary incubation with either BPD alone or BPD conjugated to an irrelevant secondary antibody, islets which underwent PDT using the specific RAMIg-BPD conjugate demonstrated elimination of APCs as assessed by immunocytochemistry. When syngeneic and allogeneic transplants were performed using islets which had undergone APC "photoablation", the histologic appearance of the grafts was compatible with either inflammation in response to non-viable tissue, or allograft rejection. The temporal disparity between the duration of tissue culture necessary to deplete islet APCs and that required to allow successful islet allografting can be variably explained. One possibility is that failure to stain APCs after a 7-10 day period of culture is not proof that these cells have been destroyed. It is conceivable that culture alters the surface of the APC such that it is no longer identified by anti-Class II MHC immunostains, but nevertheless retains its ability to present alloantigen. Alternatively, one can hypothesize that in vitro culture causes some donor tissue alteration other than APC depletion which renders it less immunogenic. The failure of PDT to permit successful syngeneic or allogeneic transplantation despite its apparent ability to eliminate islet APCs suggests that the treatment itself may cause irreversible islet injury, and that the inflammatory reaction observed is merely in response to non-viable transplanted tissue.Surgery, Department ofMedicine, Faculty ofGraduat

Twenty-year Follow-up Study of Long-term Survival of Limited-stage Small-cell Lung Cancer and Overview of Prognostic and Treatment Factors

PURPOSE: To predict the long-term survival results of clinical trials earlier than using actuarial methods and to assess the factors predictive of long-term cure in patients with limited-stage small-cell lung cancer. METHODS AND MATERIALS: Between 1981 and 1998, 1417 new cases of small-cell lung cancer were diagnosed in Saskatchewan, Canada, of which 244 were limited stage and treated with curative intent. They were followed to the end of February 2002. A parametric lognormal statistical model was retrospectively validated to determine whether long-term survival rates could be estimated several years earlier than is possible using the standard life-table actuarial method. RESULTS: The survival time of the uncured group followed a lognormal distribution. Four 2-year periods of diagnosis were combined, and patients were followed as a cohort for an additional 2 years. The estimated 10-year cause-specific survival rate was 13% by the lognormal model. The Kaplan-Meier calculation for 10-year cause-specific survival rate was 15% +/- 3%. The data also showed that the absence of mediastinal lymphadenopathy and higher chest radiotherapy dose were significant prognostic factors on multivariate analysis (p \u3c 0.05). Among the 163 patients given prophylactic cranial irradiation, a higher biologically effective dose to the brain did not improve survival or decrease the incidence of brain metastases. CONCLUSION: The lognormal model has been validated for the estimation of survival in patients with limited-stage small-cell lung cancer. A higher biologically effective dose to the brain did not improve survival or decrease the incidence of brain metastases