Oropharyngeal candidiasis may occur in up to 90% of human immunodeficiency virus (HIV)-infected patients during the course of the disease. This study is to determine the effect of prolonged highly active antiretroviral therapy (HAART) on oropharyngeal colonization with Candida species and oral candidiasis.Progressive cell-mediated immunodeficiency with decrease of CD4+ lymphocyte count to less than or equal to 200 cells/mmA prospective, longitudinal follow-up study in HIV-infected patients receiving HAART.3 and the proportion of patients whose CD4+ count less than 200 cells/mm3 decreased from 50.0% to 28.9% (p = 0.0003) in patients receiving HAART for at least 2 years. The prevalence of oral candidiasis decreased from 10.6% to 2.1% (p = 0.004). The decrease in Candida colonization was less impressive, falling from 57.8% to 46.5 % (p = 0.06). Of the 142 patients enrolled in at least two surveys, 48 (33.8%) remained colonized with Candida and 42 (29.6%) remained negative. In the remaining 52 patients, 34 switched from culture positive to negative, and an increase in CD4+ lymphocytes was noted in 91.2% of them. Among the 18 patients who switched from culture negative to positive, 61.1% also demonstrated an increase in CD4+ lymphocyte count (p = 0.01).The mean CD4+ count increased from 232.5 to 316 cells/mmCandida from the oropharynx.These findings indicate that HAART is highly effective in decreasing oral candidiasis in association with a rise in CD4+ lymphocyte counts, but only marginally effective in eliminating  Orophdidiasis ,3.Candida species. These three prospective longitudinal follow-up surveys  were designed to determine the effect of prolonged HAART on oropharyngeal candidiasis and colonization with Candida species in Taiwan.The introduction of highly active antiretroviral therapy (HAART) has led to a marked decrease in mortality and morbidity ,8 as welHIV-infected patients followed regularly at the acquired immune deficiency syndrome (AIDS) clinic at the National Taiwan University Hospital (NTUH) were enrolled in the study. The patients underwent three rounds of surveys for oral candidiasis in 1999, 2001, and 2002. A standardized case form was used to retrieve demographic information, CD4+ lymphocyte counts obtained from dates nearest to the survey culture dates and viral load. Hospitalization and receipt of antibiotics and anti-fungal drugs within three months of sampling were also recorded. For patients participated in all three surveys, the clinical data of 1999 and 2002 were used for analysis. Inclusion criteria consisted of continuous receipt of HAART, at least two complete clinical and laboratory observations during the study period and verbal informed consent. The institutional review board of the NTUH has approved the study.Cultures of the oropharyngeal and tonsillar regions were performed using a dry sponge swab  as described previously ,5. The sPlasma viral RNA load (PVL) and CD4+ lymphocyte count were usually determined at the baseline at the diagnosis of HIV infection and/or immediately before initiation of antiretroviral therapy. In those who did not initiate antiretroviral therapy, those tests were conducted on 4–6 month intervals; in those who did initiate the antiretroviral therapy, those tests were first conducted at 1 month after the beginning of the therapy, and then onto 4-month intervals regularly. When the therapy is to be changed due to virologic failure, testing protocol would be the same as those who initiated therapy for the first time. PVL were quantified using reverse transcriptase-polymerase chain reaction  with a detection limit of 400 copies/ml and CD4+ counts were determined using FACFlow (Becton Dickinson). Oropharyngeal candidiasis was characterized by painless, creamy white, plaque-like lesions on the buccal or oropharyngeal mucosa or tongue surface.All clinical and laboratory data were entered into a relational database designed for Microsoft Access 97 . The statistical significances of the differences in frequencies and proportions were determined by the Chi-square test with Mantel-Haenszel correction.3 to 316 ± 197.0 cells/mm3. The proportion of patients whose CD4+ count was ≤ 200 cells/mm3 decreased from 50.0% to 28.9 % (p = 0.0003) , 83 patients only in the 2001 and 2002 surveys (group 2), and 48 patients in all three surveys. Upon follow-up, the mean CD4+ lymphocyte count rose from 232.5 ± 180.1 cells/mmp = 0.004), while the change in prevalence of Candida colonization was less impressive, falling from 57.8% to 46.5 % (p = 0.06). Among the patients, 48 (33.8%) remained colonized with Candida and 42 (29.6%) remained negative. And in the remaining 52 patients, 34 switched from culture positive to negative alone with an increase in CD4+ cells in 91.2% of them. Among the 18 patients switching from culture negative to positive, 61.1% also demonstrated an increase in CD4+ counts. In the total 284 samplings, there were 65 with CD4+ counts ≤ 200 and were also culture-positive for Candida whereas there were 47 with CD4+ counts ≤ 200 but were culture-negative (p = 0.11). Thus, in addition to the CD4+ count, some other factors appear to have significant effects on oral colonization by Candida.In all, the prevalence of oral candidiasis decreased from 10.6 to 2.1 % (Candida and that a low CD4+ lymphocyte count (≤ 200 cells/mm3) was a risk factor for developing oral candidiasis [Candida than healthy individuals [Candida, although close to significant (p = 0.06), was not as dramatically decreased. Possible explanations include poor compliance, failure to reduce the viral load and raise the CD4+ lymphocyte count or dissociation between colonization and infection. The most likely scenario is that most patients receiving HAART continue to be colonized by Candida, but do not develop oral candidiasis.We have previously shown that hospitalization and receipt of antibiotics were risk factors for oropharyngeal colonization by ividuals . Our curividuals ,8. It waCandida. Further studies are needed to better define the relationship between Candida colonization and infection in patients with AIDS.Increase of the CD4+ lymphocyte counts is required but not sufficient to protect HIV-infected patients from colonization by HIV, human immunodeficiency virus; HAART, highly active antiretroviral therapy; AIDS, acquired immune deficiency syndrome; NYUH, National Taiwan University Hospital; PVL, Plasma viral RNA load; RT-PCR, reverse transcriptase-polymerase chain reactionThe author(s) declared that they have no competing interests.YLY designed the study with contribution with HJL and CCH. YLY drafted the manuscript. HJL conducted the experiments with contribution from YL. CCH collected swabs and patients' information.The pre-publication history for this paper can be accessed here:


To evaluate the efficacy of triple therapy consisting of single-session photodynamic therapy (PDT), intravitreal bevacizumab (IVB) and intravitreal triamcinolone (IVT) as initial pulse therapy followed by repeat intravitreal bevacizumab injections for maintenance treatment in neovascular age-related macular degeneration (AMD).In a prospective interventional case series, patients with subfoveal choroidal neovascularization (CNV) secondary to AMD underwent pulse therapy with single-session PDT according to the standard protocol followed by 1.25 mg IVB and 2 mg IVT 48 hours later. Best corrected visual acuity (BCVA) was assessed and optical coherence tomography (OCT) and fluorescein angiography (FA) were performed prior to treatment. BCVA and OCT measurements were repeated at 6 week intervals and FA was obtained after 12 weeks and when necessary thereafter. Repeat injections of IVB were performed based on fluorescein angiographic evidence of CNV leakage.This series included 17 eyes of 17 patients with mean age of 67.6 ± 7.2 years. Mean follow up duration was 50.4 ± 15.5 weeks. Mean BCVA prior to treatment was 0.74 ± 0.33 logMAR which improved to 0.52 ± 0.36 logMAR after 12 weeks (P = 0.012) and 0.41 ± 0.38 logMAR after 24 weeks (P = 0.001). Mean pretreatment central macular thickness (CMT) was 395 ± 181μ which was significantly reduced to 217 ± 69μ (P = 0.005), 231 ± 79μ (P = 0.028) and 221 ± 87μ (P = 0.05) six, twelve and twenty-four weeks after initial treatment respectively. Visual acuity improvement and CMT reduction was maintained during the follow-up period. IVB injections were repeated once, twice and three times in 10, 7 and 2 eyes at a mean interval of 20.2 ± 10.1, 19 ± 13.7 and 15 ± 1.4 weeks after initial therapy, respectively.Initial pulse triple therapy consisting of single-session PDT combined with IVB and IVT improves vision and reduces CMT in neovascular AMD. Repeat IVB injections maintain the visual gain from the initial combination therapy. Photodynamic therapy is an FDA-approved treatment for neovascular age-related macular degeneration (AMD); however this treatment modality seems to stabilize, rather than improve vision -3. FurthCombination of PDT with intravitreal injection of an anti-angiogenesis drug and a steroid may address the multifactorial pathogenesis of AMD, improve vision and reduce the need for retreatments. Results of triple therapy with PDT, bevacizumab and dexamethasone have been reported recently . We perfThe study was a prospective, interventional case series. The treatment protocol was approved by the Ethics Committee of the Ophthalmic Research Center. Patients were informed about the risks and benefits of combined therapy and written consent was obtained. Eyes with all types of active subfoveal CNV secondary to neovascular AMD with lesions less than 4 disc areas and Snellen visual acuity of 20/30 to 20/400 at baseline were included. Exclusion criteria were glaucoma, diabetic retinopathy, macular disorders other than AMD and previous treatment for CNV.Eligible eyes were evaluated before treatment completely. Best corrected visual acuity measured by Snellen chart was recorded in logMAR. Applanation tonometry, anterior segment examination with slit lamp and fundus examination was performed. All eyes underwent color fundus photography and fluorescein angiography (FA) using a confocal scanning laser angiograph (Heidelberg Retina Angiograph II: Heidelberg engineering). Optical coherence tomography  was performed before intervention for all cases to measure the central macular thickness (CMT). Photodynamic therapy with verteporfin was performed according to the standard regimen. Forty eight hours after PDT, intravitreal injection of 1.25 mg/0.05 ml bevacizumab  and 2 mg/0.05 ml triamcinolone acetonide was carried out at two different sites under sterile condition. A topical antibiotic, every 6 hours for 3 days, was prescribed and patients were instructed to return in case of ocular pain or redness or any deterioration of vision. Patients were examined one day and one week after injection. Topical antiglaucoma medication was prescribed if intraocular pressure (IOP) was more than 21 mmHg.Visual acuity and optical coherence tomography (OCT) were evaluated at 6 week intervals. Fluorescein angiography was repeated at week 12 and when considered necessary thereafter. CNV activity was defined as presence of leakage from the CNV on fluorescein angiography. Eyes with active CNV underwent repeat intravitreal injection of 1.25 mg bevacizumab. Repeat PDT and intravitreal triamcinolone injections were not considered in the study protocol.Paired T-test was used to compare findings before and after treatment.Seventeen eyes of 17 patients  with the mean age of 67.6 ± 7.2 years were included in this study. Lesion types were predominantly classic in 5 eyes, minimally classic in 3 eyes, occult in 8 eyes and retinal angiomatous proliferation (RAP) in one eye. Mean follow-up was 50.4 ± 15.5 weeks and all 17 patients completed the 24 week follow up (Table Mean BCVA was 0.74 ± 0.33 logMAR range: 1.20 to 0.22) before treatment (Table 0 to 0.22Seven eyes (41.2%) remained stable following the initial therapy. Follow up period for these 7 eyes ranged from 24 to 73 weeks (mean: 42.6 ± 17.6 weeks). A second intravitreal injection of bevacizumab was required in 10 eyes (58.8%), a third injection was performed in 7 eyes (41.2%) and a fourth injection was needed in 2 eyes (11.8%). Mean interval between the first and second injections and between the second and third injections were 20.2 ± 10.12 and 19 ± 13.7 weeks respectively. The interval between the third and fourth injections had a range of 14 to 16 weeks. Mean follow up for eyes with 2, 3 and 4 injections were 56 ± 11.6 weeks, 60.1 ± 5.5 weeks and 58.5 ± 9.2 weeks respectively. No adverse ocular or systemic events were observed during the follow up period.Mean IOP was 14 ± 1.9 mmHg before treatment and increased to 17.3 ± 6.3 mmHg at week 6 and returned to pretreatment levels 12 weeks after intervention. In one patient IOP was increased to 34 mmHg after one week. Intraocular pressure was controlled in this patient with a combination of timolol eye drop bid and dorzolamide eye drop every 8 hours, which were continued for 5 weeks.In this small case series, combination of single-session PDT with intravitreal bevacizumab and triamcinolone was used as pulse therapy for neovascular AMD. Our goal was to increase the efficacy of treatment and reduce side effects of monotherapy. Triple therapy resulted in significant visual improvement and CMT reduction irrespective of lesion type and also reduced the need for retreatments. Visual acuity improved significantly in the majority of cases in our series and remained unchanged in others. This treatment effect persisted during the follow up period. More than one third of our patients remained stable after the initial pulse therapy. The visual gain could be maintained in the remaining cases with few retreatments. Intravitreal bevacizumab was used for maintenance therapy to avoid the side effects of repeat PDT and repeat intravitreal triamcinolone. The average interval between the first and second treatment sessions was about 20 weeks which may reflect less need for retreatment following the initial pulse therapy.Augustin et al recently published the results of triple therapy with PDT, bevacizumab and dexamethasone for neovascular AMD . They coThe rationale for single-session PDT emanates from the present knowledge regarding the possible adverse effects of repeat PDT on physiological choroid and even RPE and neurosensory retina ,25. In aPDT may cause phototoxicity-induced VEGF expression and increase vascular permeability by triggering generation of free radicals and lipid peroxides . TriamciWe used intravitreal bevacizumab as one of the components of pulse triple therapy. It was also used as a maintenance therapy in our series. Use of an antiangiogenic factor can counteract the PDT induced over-expression of VEGF and reduce its adverse effects. On the other hand, PDT may disrupt the architecture of CNV and help the antiangiogenic factor to affect the lesion more potently . Aggio eThe optimal time for intravitreal injection of bevacizumab with or without a steroid after PDT is not known. It has ranged from 16 hours to 2 weeOur preliminary results suggest that intravitreal bevacizumab has a synergistic effect with PDT and intravitreal triamcinolone for treatment of neovascular AMD. Using this triple therapy as an initial pulse can improve or stabilize visual acuity. In case of recurrent CNV, repeat intravitreal injection of bevacizumab may help maintain the visual gain. Further studies are required to confirm the results of this pilot study.LogMAR: Logarithm of the Minimum Angle of ResolutionVEGF: Vascular Endothelial Growth FactorRPE: Retinal Pigment EpitheliumThe author(s) declare that they have no competing interests.HA participated in the design of the study, contributed in acquisition and analysis and interpretation of data, was involved in drafting the manuscript and its revising. RT participated in the design of the study, contributed in acquisition and analysis and interpretation of data, was involved in drafting the manuscript and its revising. MS participated in the design of the study, contributed in acquisition and analysis and interpretation of data, gave final approval of the version. MR participated in the design of the study, contributed in acquisition and analysis and interpretation of data, gave final approval of the version. RK participated in the design of the study, contributed in acquisition and analysis and interpretation of data, gave final approval of the version. AL participated in the design of the study, contributed in acquisition and analysis and interpretation of data, gave final approval of the version. MA participated in the design of the study, contributed in acquisition and analysis and interpretation of data, gave final approval of the version. MD participated in the design of the study, contributed in acquisition and analysis and interpretation of data, gave final approval of the version. SM participated in the design of the study, contributed in acquisition and analysis and interpretation of data, gave final approval of the version.The pre-publication history for this paper can be accessed here:


Inhaled mannitol is a new bronchial provocation test (BPT) developed to improve portability and standardisation of osmotic challenge testing. Osmotic challenge tests have an advantage over the traditional methods of measuring airway hyperresponsiveness using methacholine as they demonstrate higher specificity to identify asthma and thus the need for treatment with inhaled corticosteroids (ICS). The safety and the efficacy of mannitol (M) as a BPT to measure airway hyperresponsiveness were compared to hypertonic (4.5%) saline (HS) in people both with and without signs and symptoms of asthma.1 was measured 60 seconds after each dose of mannitol  and after each exposure to HS . A 15% fall in FEV1 defined a positive test. Adverse events were monitored and diaries kept for 7 days following the tests.A phase III, multi-centre, open label, operator-blinded, crossover design, randomised trial, with follow-up. Asthmatics and non-asthmatics (6–83 yr) were recruited and 592 subjects completed the study. Mannitol was delivered using a low resistance dry powder inhaler and HS was delivered using an ultrasonic nebuliser. The FEVMean pre-test FEV1 (mean ± SD) was 95.5 ± 14% predicted. 296 were positive to mannitol (M+) and 322 positive to HS (HS+). A post study physician conducted clinical assessment identified 82.3% asthmatic (44% classified mild) and 17.7% non-asthmatic. Of those M+, 70.1% were taking ICS and of those mannitol negative (M-), 81.1 % were taking ICS. The % fall in FEV1 for mannitol in asthmatics was 21.0% ± 5.7 and for the non-asthmatics, 5.5% ± 4.8. The median PD15 M was 148 mg and PD15 HS 6.2 ml. The sensitivity of M to identify HS+ was 80.7% and the specificity 86.7%. The sensitivity of M compared with the clinical assessment was 59.8% and specificity 95.2% and increased to 88.7% and 95.0% respectively when the M- subjects taking ICS were excluded. Cough was common during testing. There were no serious adverse events. The diarised events were similar for mannitol and HS, the most common being headache , pharyngolaryngeal pain , nausea , and cough .The efficacy and safety of mannitol was demonstrated in non-asthmatic and clinically diagnosed asthmatic adults and children. The t.5% salinormality . The dosss (AHR) . There aIn order to overcome some of these problems a dry powder preparation of mannitol was developed to provide a more simple form of osmotic challenge . The stuWe aimed to determine the efficacy and safety profile of a commercial preparation of dry powder mannitol in subjects with and without signs and symptoms of asthma. A phase III, multi-centre, open label, operator-blinded, crossover design, randomised trial, with follow-up was performed comparing the airway response to mannitol with the HS test and a standard clinical assessment of asthma.The study was an operator blinded, randomised, crossover comparator study. At Visit 1, after entry and randomisation to determine the order of each challenge, each subject answered a questionnaire, had skin prick tests for aeroallergens then underwent the first challenge. The second challenge was scheduled one week later at Visit 2 and the study was complete a week after the second challenge at Visit 3, when each subject returned for a measurement of spirometry. The respiratory scientist conducting the initial challenge was blinded to the subject's asthma status and the respiratory scientist conducting the reciprocal challenge was blinded to the results of the initial challenge and the asthma status of the subject. The respiratory physician was blinded to the results of the mannitol challenge and the identity of the subject when determining the asthma status of the subject.The protocol was approved by the Ethics Review Committee of each participating hospital. The trial was carried out under the Clinical Trial Notification scheme of the Therapeutic Goods Administration of Australia .Of six hundred and fifty-four subjects 6 years of age and above recruited from the local community and from pulmonary function clinics, 646 completed at least one challenge and were included in the population that could be analysed for the safety analysis Table  Figure . Of thesThe asthmatic subjects were required to have active signs and symptoms of asthma according to the National Asthma Council of Australia Asthma Management Handbook Guidelines . The non1 greater than 70% of normal predicted values for asthmatic subjects OR greater than 80% of normal predicted values for non-asthmatic subjects. No subject was to have an active upper or lower respiratory tract infection severe enough to require a medical consultation or any other acute or chronic pulmonary disorder including:- cystic fibrosis, chronic obstructive pulmonary disease, bronchiectasis, chronic bronchitis, emphysema, tuberculosis, and carcinoma. Further, subjects with current uncontrolled hypertension or known aortic aneurysm, myocardial infarction or cerebral vascular accident in the last six months, or ocular or abdominal surgery in the three months prior to enrolment were excluded from the study.Subjects were required to have a baseline FEVNo subject could be breast feeding or pregnant, or have participated in another trial in the previous 4 weeks, be related to a study investigator, or have a known intolerance to mannitol or salbutamol. No subject was to have a previous admission to Intensive Care for asthma in the two years prior to study entry, and no subject could be taking oral / parenteral corticosteroids in the two weeks prior to study enrolment.1 was required to be within 10% of that at Visit 1. Challenges were performed at the same time of day ± 2 hours.All subject were required to be able to perform repeatable spirometry according to American Thoracic Society (ATS) criteria , and to Two questionnaires were carried out, one investigating respiratory history over the preceding 12 months and based on the International Study of Asthma and Allergies in Childhood (ISAAC) Steering Committee Phase 1 and 2 modules . The othEach subject was issued with a study diary at Visit 1 and requested to complete the diary every day up to their third visit when they handed it to the investigator. The diary was a record of adverse events, respiratory symptoms and concomitant medications. The investigator was responsible for reviewing the diary for completeness at the final visit.1 to be measured 60 seconds after each mannitol dose . If the FEV1 fell by = 10% on any one dose then that same dose was repeated.Dry powdered mannitol (Aridol™) was supplied in kit form  and contained one empty capsule (0 mg), 2 × 5, 2 × 10, 2 × 20, and 18 × 40 mg capsules. The dry powder device used for inhalation was the Osmohaler™ , a single capsule device with a low inspiratory resistance. The mannitol challenge required the FEV1, the inclusion of the 0 mg capsule demonstrated the sound and use of the device to the subject. Hearing the capsule rotating indicated that sufficient inspiratory flow had been achieved and that it was positioned correctly in the chamber. Sixty seconds after inhalation of the 0 mg capsule the FEV1 was measured and two repeatable values were obtained and recorded. The highest of these values was taken as the baseline FEV1 and was used to calculate the target FEV1 value that indicated a 15% fall in response to the mannitol challenge. This value was calculated immediately after the administration of the 0 mg capsule.The challenge started with the empty capsule. This was loaded into the device and punctured by the investigator. The subjects were asked to inhale from the device from near to functional residual capacity to near to total lung capacity and to hold their breath for 5 seconds. Subjects were encouraged to keep a nose clip on for 10 seconds after inhalation and then exhale through their mouth to minimise deposition in the nasopharynx. In addition to providing the baseline FEV1 was measured to obtain two repeatable FEV1 values. This procedure was repeated for each dose step until a 15% fall in FEV1 was achieved or the cumulative dose of 635 mg had been administered. At each dose step, oxygen saturation and cough severity were recorded.The first dose of mannitol (5 mg) was administered, and sixty seconds later the FEV1 was measured at least twice 60 seconds after each exposure time. The subjects wore a nose clip and were encouraged to breathe normally through a two-way non-rebreathing valve. If the fall in FEV1 was = 10% on any one exposure time the same exposure time was repeated. The ultrasonic nebuliser was required to have an output of at least 1.5 ml/min. After each exposure time, oxygen saturation and cough severity were recorded.The initial exposure time for inhaling the 4.5% saline was for 30 sec, then 1 min, 2 min, 4 min, and 8 min. The FEV1 measurements made after each dose was used in the calculation for the % fall in FEV1. The dose of mannitol (mg) or saline (ml) to provoke a 15% fall in FEV1 (PD15) was calculated by linear interpolation from the curve relating the % fall in FEV1. For mannitol this was from the post 0 mg capsule baseline value for FEV1 to the cumulative dose of mannitol delivered . For saline, the amount of aerosol delivered per minute was calculated by dividing the total amount delivered by the time of delivery (e.g. 28 ml in 15.5 min = 1.81 ml/min). The dose was expressed cumulatively over time.The highest value of 2 repeatable FEV1 recorded and dividing it by the cumulative dose of mannitol or saline administered to induce that % fall. For calculation of the RDR a value of 0.1 was used for those having 0% fall in FEV1.The response dose ratio (RDR) was calculated by taking the final % fall in FEV1 from baseline occurred. Subjects who reached the end of a challenge with <15% reduction in FEV1 were considered to have a negative response.Efficacy was analysed using sensitivity and specificity of the mannitol challenge with respect to the 4.5% saline challenge and the clinical assessment. Subjects were considered positive to a test if at least a 15% reduction in FEV1 received 200 mcg of the beta2 adrenoceptor agonist salbutamol via a Volumatic™ spacer. If the FEV1 had not returned to within 5% of the baseline FEV1 in 15 minutes a second dose of salbutamol was administered.On completion of the challenge, subjects with a 15% or more percentage fall in FEV2), respiratory rate, and heart rate measured at the beginning of the challenge, on immediate completion of the challenge , and 15 minutes after challenge.Vital Signs measured were sitting blood pressure, oxygen saturation by pulse oximetry  in subjects that had both mannitol and HS challenge, and in terms of the investigator's clinical assessment or the classification of asthmatic/symptomatic or non-asthmatic at study entry. The challenge results are represented as: M+/M- = positive/negative test result with mannitol, S+/S- = positive/negative test result with HS, A+/A- = asthmatic-symptomatic/non-asthmatic at study entry, C+/C- = asthmatic/non-asthmatic by physician's clinical diagnosis at Visit 3. Cross-tabulations were performed to create 2 × 2 tables of the number of subjects within each of four cells for each challenge. In the analysis of mannitol vs HS, the four cells comprised of those positive to both challenges , those negative to both challenges , those positive to mannitol and negative to HS , and those negative to mannitol and positive to HS . Sensitivity was calculated as the probability of a positive test result with mannitol, given a positive HS result (Pr(M+ | S+)), i.e. the number of subjects positive to mannitol out of the total number of subjects positive to HS. Specificity was calculated as the probability of a negative test result with mannitol, given a negative HS result (Pr(M- | S-)), i.e. the number of subjects negative to mannitol out of the total number of subjects negative to HS.A cross over logistic regression analysis was performed using the results of the mannitol and HS challenges to examine sequence effects, and another to look for study site differences. A cross over regression analysis was also performed to investigate differences between mannitol and HS in the change from baseline  to end of challenge for the vital sign parameters: systolic and diastolic blood pressure, oxygen saturation, heart rate and respiratory rate.1 values and there was no difference compared to the non-asthmatics  that included both asthmatics and non-asthmatics, the severity of symptoms using the questionnaire specific to the week prior to commencement of the study revealed that there were 39.0% (n = 231) with wheeze, 32.8% (n = 194) with trouble breathing, and 11.3% (n = 67) who had respiratory symptoms that interfered with normal activities. There were 45.2% (n = 292) who had daytime cough that lasted for a mean ± SD of 4.0 ± 2.5 days, with 82.5% (n = 241) stating that it was only occasional. Cough at night time was reported by 31.6% (n = 187) and 49.5% (n = 293) reported using a reliever puffer in the past week. Only 6.3% (n = 37) reported having a cold or flu in the past week. There were 78.4% taking at least one medication for asthma. Of those assessed as asthmatics at entry 27.7 % were taking ICS alone and 38.5% in combination with a LABA, 15.6% were taking only a SABA and 3 subjects taking only a LABA. Only 1.7% were taking leukotriene antagonists, 4.6% were taking an anti-cholinergic and 2.2% were taking sodium cromoglycate.As designated by the clinical assessment, 82.3% (n = 487) of the efficacy population (n = 592) were asthmatic and 17.7% (n = 105) were non-asthmatic. Of the 378 adult asthmatic subjects, 50.5% (n = 191) were classed as mild, 39.9% (n = 151) were classed as moderate, and 9.5% (n = 36) as severe . The maximum mean % fall in FEV1 in children less than 12 years of age was 21.1% ± 6.0 (range:15.85 × 40.30) for mannitol and 22.0% ± 6.4 (range:16.34–48.62) for HS. Only 14 subjects recorded a maximum fall in FEV1 greater than 30% and the majority had a fall between 15% and 25%  were positive to the mannitol challenge and 54.4% (n = 322) positive to HS. For those who were positive the maximum mean % fall in FEV1 was similar for both mannitol (21.0% ± 5.7 (range: 15.0–45.5)) and for HS (21.3% ± 5.9 (range:15.0–48.62)) and there was a relationship between maximum % fall in FEV15 was 112.20 mg  for mannitol and 5.01 ml,  for HS. The median PD15 for mannitol was 148.1 mg and for HS it was 6.2 ml. There was a moderate, though statistically significant, correlation between log transformed PD15 between mannitol and HS . Those adults with a classification of mild asthma had a PD15 for mannitol that was significantly different from those classed as having moderate and severe asthma  for PD1 from baseline for subjects who were clinically assessed as non-asthmatic was 5.5% ± 4.8 after mannitol and 5.4% ± 4.9 after HS. The median (range) RDR for those with a negative challenge was 0.0075 (0.0–0.0317) for mannitol and 0.15 (0.0–0.70) for HS. For the group who were non-asthmatic the RDR geometric mean (95% CI) for mannitol was 0.0029  and for the HS was 0.11 .The maximum mean % fall in FEVThe highest proportion (28.7%) of subjects achieving a positive mannitol challenge result did so in the cumulative dose range of >155 × 315 mg, 18.2% were positive in the >75 × 155 dose range and 16.2% in the >35 × 75 dose range  and no significant difference between the treatment order or the study site for mannitol and HS responses.Nineteen (3.0%) subjects had repeat mannitol doses, 89.5% (n = 17) of whom went on to achieve a PDThe mean ± SD time taken for a positive mannitol challenge was 17.3 ± 7.09 minutes and for HS was 15.0 ± 9.1 minutes. For a negative challenge the mean ± SD for mannitol was 26.4 ± 6.1 minutes and for HS it was 27.3 ± 3.6 minutes. It should be noted that this included the time taken to complete the post challenge measurement of the vital signs.1 was similar for both mannitol and HS, being 19.4 ± 8.8 minutes and 19.3 ± 8.7 minutes respectively. The maximum recovery time was 65 minutes for mannitol and 79 minutes for HS. There were 344 (54.9%) subjects who received 200 mcg of salbutamol after the mannitol challenge, and 370 (58.2%) after HS. A second salbutamol dose of 200 mcg was given to 46 (7.3%) subjects after mannitol and 38 (6.0%) after HS, with additional bronchodilator given in 6 (1.0%) subjects following the mannitol challenge and 2 (0.3%) following HS.For subjects with a positive challenge test, the time to recover to 95% baseline FEV1 at the time they were stopped.There were 535 (85.3%) subjects who experienced cough during the mannitol challenge and 468 (73.5%) during the HS challenge. The mean cumulative dose at which cough was first reported was 62.7 mg for mannitol and 5.07 ml for HS. The majority of subjects, 70.8% for mannitol and 64.8% for HS, experienced occasional cough causing no delay in administering the next dose. Frequent cough causing a delay in the challenge was noted in 83 (13.2%) subjects during the mannitol challenge and 51 (8%) subjects during HS. There were 8 (1.3%) subjects during mannitol and 5 (0.8%) during HS who were noted to have a cough severe enough to stop the challenge although 2 and 4 respectively did record a 15% fall in FEV1  and cough (see above).There were 14 (2.2%) subjects who commenced but did not complete the mannitol challenge and 9 (1.4%) subjects who did not complete the HS challenge. The most frequent causes of an incomplete challenge were incorrect calculation of the target 15% reduction in FEVThe sensitivity and specificity for mannitol to identify responsiveness to HS and for the clinical diagnosis are given in Table 2 agonist) were divided into mannitol positive and negative. Of the 291 subjects mannitol positive, 204 (70.1 %) had been using ICS prior to the first challenge; of the 196 subjects negative to mannitol, 159 (81.1%) had been using ICS prior to the first challenge. As an exploratory analysis, the subjects who were mannitol negative and taking ICS before the study were removed from the analysis of sensitivity and specificity for mannitol with respect to the clinical assessment and there was an improvement in sensitivity (88.7%) and specificity (95.0%).Subjects diagnosed with asthma who were using ICS  reported adverse events after mannitol and 44.7% (n = 284) after the HS. Applying the MedDRA classifications  after mannitol and 4.7% (n = 30) after HS. The most common were respiratory, thoracic and mediastinal disorders reported in 2.1% (n = 13) after the mannitol and 1.4% (n = 9) after HS. Nervous system disorders were reported in 1.6% (n = 10) after mannitol and 1.9% (n = 12) after HS. These were headache in 1.3% (n = 8) after mannitol, and 1.1% (n = 7) after HS, followed by cough or aggravated cough in 0.8% (n = 5) after mannitol, and 0.3% (n = 2) after HS.Withdrawal from the study due to adverse events occurred in four (0.6%) after the mannitol, and three 0.5%) after the HS. There were no statistically significant carryover effects for any of the baseline, end challenge, or recovery values for systolic and diastolic blood pressure, oxygen saturation, heart rate, and respiratory rate, either at the end of challenge or at recovery (p > 0.2)  and HS (6.1 ml) [The median doses of mannitol (148 mg) and HS (6.2 ml) to cause a 15% fall in FEV(6.1 ml) . Thus 50These results demonstrate the safety of the inhaled mannitol challenge to identify AHR. There were no serious adverse events recorded. There was no difference in the overall safety profile demonstrated by a similar adverse event profile between mannitol and HS. The low incidence of headache, the most frequent complaint after both challenges, was likely to be a direct result of the challenge procedure. Headache was more prevalent in those with a negative mannitol challenge and likely due to the larger number of forced expiratory manoeuvres required compared to those with a positive challenge.1 was similar for both the mannitol and HS challenges and close to the target FEV1 of 15%. Relative to the potential fall in FEV1 that can occur following challenges such as exercise, this is a moderate decrease in FEV1. Few subjects had a fall in FEV1 greater than 30%, a value that is frequently attained when testing with exercise or with dry air as the stimulus [1 were 15% and 26%. Twelve subjects had a PD15 to hypertonic saline = 1.0 ml which is equivalent to a 30 sec exposure to the aerosol.The mean maximum fall in FEVstimulus ,29. Only1 of >10% failed to achieve a PD15 and for both these subjects the maximum fall achieved was greater than 14.5%. Thus a fall in FEV1 of 10% between doses of mannitol should also be considered as a positive challenge and signal an end to the challenge procedure. The PD15 can then be extrapolated from the recorded response at the 10% fall.Only two of the 19 subjects who had a between dose fall in FEV2, e.g. exercise challenge [2 during the mannitol challenge was small and was probably of clinical significance in only 3 subjects . There were only small and clinically insignificant changes in blood pressure, heart rate, and breathing frequency after challenge. This is in contrast to a challenge with exercise that can be associated with substantial changes in blood pressure, heart rate, and breathing frequency [Bronchoconstriction provoked by other challenge tests may be accompanied by significant reductions in SpOhallenge . Howeverrequency . The insCough is a recognised aspect of both the mannitol and HS challenges . The maj15>6.1 ml). The reason for the discrepancy is not clear from the results but several explanations can be postulated. One explanation is that the HS is administered with tidal breathing whereas mannitol is given with an inhalation to total lung capacity. Cockcroft et al [The sensitivity of mannitol to identify subjects with AHR to HS was lower than expected, given the results of an earlier study . The majft et al  have repft et al . Other pIt is likely that the relatively high number of negative challenges in subjects with asthma is largely due to the fact that such a high proportion of the subjects were using ICS and therefore their airway inflammation was well controlled. Airway sensitivity to mannitol is reduced or inhibited by treatment with ICS ,18, a feBased on earlier studies, the sensitivity of the mannitol challenge would have been expected to be higher and, up to 95% in asthmatics not taking ICS . Of the Koskela et al  have sug15 had a sensitivity of 81% and specificity of 87% with respect to PD15 for 4.5% saline. A positive mannitol test cut off of a 15% fall in FEV1 (PD15) provided appropriate sensitivity and specificity with respect to clinical diagnosis of asthma even when the patient's baseline FEV1 was within the normal range. Based on an analysis of patients with a clinical diagnosis of asthma, and excluding those with a negative test result and on current corticosteroid therapy, mannitol PD15 had a sensitivity of up to 89% to detect the presence of asthma and specificity of 95% for clinical diagnosis of asthma.In conclusion the mannitol challenge was generally safe and well tolerated. No serious adverse events were recorded. Mannitol PDThe study reported in this paper was sponsored by Pharmaxis Ltd. BC, AL and RF-M are shareholders, have options and are employees of Pharmaxis Ltd.At the time of the study Dr Anderson, Dr Brannan, Ms Perry and Ms Freed-Martens were all employed, by the Central Sydney Area Health Service  who owns the patents on the application described here for mannitol. All employees have benefited from the monies arising from the study in terms of attendance at scientific meetings but they have not received a personal financial benefit. Dr Anderson is the inventor named on the patent. The Area Health Service licensed the intellectual property to Pharmaxis Ltd in 2001.The authors have no non-financial competing interests.All the authors contributed to the design of the protocol for this study. The data management and statistical analysis of the results was contracted out by Pharmaxis Ltd to Datapharm Australia Pty Ltd. This paper is a summary of their report and all the authors made a contribution to this summary.


Fibrolamellar Carcinoma (FLC), a subtype of hepatocellular carcinoma (HCC), is a rare primary hepatic malignancy. Several aspects of the clinic features and epidemiology of FLC remain unclear because most of the literature on FLC consists of case reports and small cases series with limited information on factors that affect survival.We did a retrospective analysis of the clinical and histological characteristics of FLC. We also determined the rate of cellular proliferation in biopsies of these tumors. We assessed whether these variables were associated with survival.We found 15 patients with FLC out of 174 patients with HCC (8.6%). Between patients with these neoplasms, we found statistically significant survival, age at onset, level of alpha fetoprotein, and an earlier stage of the disease. The 1, 3 and 5 year survival in patients with FLC was of 66, 40 and 26% respectively. The factors associated with a higher survival in patients with FLC were age more than 23 years, feasibility of surgical resection, free surgical borders, absence of thrombosis or invasion to hepatic vessels and the absence of alterations in liver enzymes. The size of the tumor, gender, cellular proliferation and atypia did not affect the prognosis.We concluded that FLC patients diagnosed before 23 years of age have worse prognosis than those diagnosed after age 23. Other factors associated with worse prognosis in this study are: lack of surgical treatment, presence of positive surgical margins, vascular invasion, and altered hepatic enzymes. Hepatocellular carcinoma (HCC) is the most frequent primary neoplasm of the liver. Its incidence is increasing in western countries mainly due to the growing Hepatitis C Virus epidemics. Though potentially curable with surgery, very few patients are candidates for resection because of tumor extension and poor liver function at diagnosis. The prognosis is poor, with a 1-year survival rate of 22.6% [Fibrolamellar carcinoma (FLC) was described initially in 1958 by Edmonson, as a subtype of HCC . It is cThe differences in the prognosis of these patients are probably a consequence of a non cirrhotic liver and the expanding nature tumor growth permits a high rate of surgical resection. In contrast to classic HCC, which has well defined prognostic factors such as Alfa fetoprotein levels, performance status, liver function, and the severity of cirrhosis -14, no sMedical records of patients with the diagnosis of HCC were reviewed in the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán from 1990 to 2003. Patients with FLC were then identified from biopsy results. Patients with incomplete medical records or lacking biopsy results were excluded from the study.Biopsies of patients with FLC were reviewed to confirm the diagnosis; some samples where dyed again to determine histopathologic characteristics such as atypia and mitosis index. Additional samples were analyzed by immunohistochemistry with the avidin-biotin-peroxidase complex and counterstained with hematoxylin; they were incubated overnight at 4°C either with rabbit anti-human proliferation cell nuclear antigen as a marker of cellular synthesis phase. A pathologist of our institution, blinded to the results of survival, counted the number of cell nuclei positive for nuclear antigen found per microscopy field at × 40 magnification in 10 different fields .t test and the chi square or Fisher's exact test was used to compare the continuous variables with normal distribution and nominal variables of patients with classic HCC and FLC respectively. Kaplan-Meier curves and log-rank was used for the survival analysis. The multivariate analysis was done using Cox regression modelling.The variables of survival, alpha fetoprotein, mitotic index and cellular proliferation index were expressed as mean ± standard error. The SPSS program version 10 was used for the statistic analysis. Statistic significance was determined as P < 0.05 with two-sided test. The Student's We reviewed 174 medical records of patients with diagnosis of HCC and found 15 patients with FLC,(8.6%). Seventy percent of the FLC patients were females compared to 37% of the classic HCC patients (p = 0.045). The average age was lower in patients with FLC compared to patients with HCC (23 ± 2.6 years with a range of 17–45 years vs 58 ± 1.5 years with a range of 22–87 years respectively p = 0.001). No patient with FLC had a previous history of blood transfusions or a positive serology for hepatitis. In contrast, 75% of patients with HCC had positive hepatitis B and/or C virus serology (p = 0.0001). Thirty percent of the patients with FLC reported use of oral contraceptives, with a range of use of 6–60 months, versus 15% of the patients with classic HCC (p = 0.1). Most common signs and symptoms in patients with FLC were: right upper quadrant pain 83%, hepatomegaly 75%, and weight loss 67%. In laboratory screening we found mean serum albumin of 3.05 ± 0.2 vs 2.9 ± 0.1 g/dL in patients with classic HCC (p = 0.8). The alpha fetoprotein levels were significantly lower in patients with FLC compared to patients with classic HCC . The patients with FLC and classic HCC presented stage I 27% vs 6% (p = 0.026), stage II 7% vs 16% (p = 0.46), stage III 40 vs 67% (p = 0.042) and stage IV 27% vs 10% (p = 0.1) respectively. The mean survival time of patients with FLC was 30 ± 6.4 months vs 10.6 ± 3 of the classic HCC (p = 0.007) , lymphadenopathy (14%), linear hypodense images corresponding to central scar (13%), peripheral reinforcement (12%). The MRI characteristics were central scar (40%), defined contour (18%), more than one tumor (12%), and multilobular contour (12%).Histopathologic characteristics of FLC biopsies included absence of cirrhosis, presence of fibrotic septum and granular cytoplasm. Furthermore, 92% of the patients demonstrated polygonal cells. Also, 75% of the patients had mild atypia with the remainder showing moderate atypia. In addition, 42% of the biopsies demonstrated pseudoinclusions; 42% had prominent nucleolus, while 33% showed hepatic steatosis Figures  and 2B. Surgical resection was performed in 80% of the FLC patients. The most frequent procedures were exploratory laparotomy with biopsy (40% of the patients), lobectomy (20%) and segmentectomy (20%). 60% had free surgical margins and 43% percent received chemotherapy. The overall survival of patients with FLC at 6, 12, 24, 36 and 60 months was 66, 66, 53, 40 and 26% Figure  and normFactors unrelated to prognosis were gender (p = 0.646), presence of palpable mass (p = 0.84), hepatomegaly (p = 0.68), weight loss (p = 0.64), ascites (p = 0.57), fever (p = 0.18), jaundice (p = 0.34), hemoglobin value (p = 0.52), normal alkaline phosphatase (p = 0.68), hypoalbuminemia (0.9), increase in alpha fetoprotein (p = 0.67), level of atypia (p = 0.31), mitotic index (p = 0.9), index of cellular proliferation (p = 0.8) and TNM stage (p = 0.69). Multivariate analysis showed only age greater than 23 years (p = 0.027) and absence of multiple tumors in more than one lobe (p = 0.043) were associated with better survival.Fibrolamellar Hepatocarcinoma is a rare primary hepatic malignancy constituting approximately 1% of all cases of primary liver cancer. A recent retrospective study, the Surveillance, Epidemiology and End Results (SEER) program diagnosed only 68 cases of FLC out 7,896 cases of HCC (0.86%); nevertheless, FLC made up 13.4% of all HCC tumors in people below the age of 40 years . In our Another interesting issue was the higher frequency of female gender in the group of FLC compared to classic HCC (60 vs 37%). This observation was also reported in the SEER study where the authors found a higher proportion of females (51.5 vs 26.3%) . Some caWe found a significantly lower age at diagnosis in patients with FLC, similar to results reported in the SEER study; the age for FLC at diagnosis was 39 ± 20 vs 65 ± 13. A greater proportion of patients with FLC were classified as having localized disease (stage I) compared to patients with HCC. Similar results were described by the SEER study, where localized disease at diagnosis was seen in 41.2% of FLC patients vs 30.9% of HCC patients. One of the main radiological features found in our study was the presence of central scar. A retrospective study analyzed different radiological features in this kind of tumors and found central scar 71% on CT scans and 82% on MRI in patients with FLC .A limitation of the SEER study was the absence of analysis of factors that modified survival. We found important differences in survival related to age; younger patients (less than 23 years) had a worse prognosis than those who were older. Another associated factor was the feasibility of surgical resection (multiple tumors in more than one lobe) and the presence of tumor in, and thrombosis of, liver vessels independent of the tumor size. We found that histological characteristics such as atypia and cellular proliferation index did not influence prognosis.The marked differences in epidemiology and clinical course indicate that FLC is distinct from HCC. We found that patients with FLC had higher survival rates than patients with HCC. These results are consistent with the published literature. Some authors report a higher survival rate in patients with FLC after surgical resection, compared to patients with HCC in absence of cirrhosis . In the We concluded that FLC patients diagnosed before 23 years of age have worse prognosis than those diagnosed after age 23. Other factors associated with worse prognosis in this study are: lack of surgical treatment, presence of positive surgical margins, vascular invasion, and altered hepatic enzymes.The author(s) declare that they have no competing interests.JGL and AT reviewed medical records and participated in the design BM reviewed the pathological material and helped to draft the manuscript. MU and ELR participated in the design of the study and coordination. LML participated in the design of the study and performed the statistical analysis. OA conceived of the study, and participated in its design and coordination, performed the statistical analysis and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


A case control study on patients with soft tissue sarcoma and malignant lymphoma was undertaken to test whether there was any association between these diseases and past exposure to chlorinated phenoxy acid herbicides or chlorophenols. It was carried out over the period 1982-1988 in Victoria, Australia. Thirty males with soft tissue sarcoma and 52 males with malignant lymphoma were matched by age, place of residence and sex with one population control and one cancer control each. Exposure was assessed by personal interviews conducted by an occupational hygienist. Exposures within 5 years prior to diagnosis of each matched case were ignored, both for the cases and their matched controls. The estimated relative risks for definite or probable exposure to chlorinated phenoxy compounds or chlorophenols for at least 1 day were 1.0 : 0.3-3.1) for soft tissue sarcoma and 1.5 (95% CI: 0.6-3.7) for malignant lymphoma. When the criterion for exposure was raised to more than 30 days, the estimated relative risks were 2.0 (95% CI: 0.5-8.0) for soft tissue sarcoma and 2.7 (95% CI: 0.7-9.6) for malignant lymphoma. Additional analyses were carried out for exposure of at least 1 day to phenoxy herbicides alone or chlorophenols alone. None of the estimated relative risks was significantly greater than unity.


The UK government proposed introducing partial smokefree legislation for England with exemptions for pubs and bars that do not prepare and serve food. We set out to test the hypothesis that pubs from more deprived areas and non food-serving pubs have higher levels of particulate air pollution.2.5) levels over a minimum of 30 minutes in areas where smoking was allowed,, and calculated mean time-time weighted average PM2.5 levels.We conducted a cross sectional study in four mainly urban areas of the North West of England. We recruited a stratified random sample of 64 pubs divided into four groups based on whether their local population was affluent or deprived (using a UK area based deprivation measure), and whether or not they served food. The timing of air quality monitoring stratified to ensure similar distribution of monitoring by day of the week and time of evening between groups. We used a portable air quality monitor to collect fine particle (PM2.5 was 285.5 μg/m3 (95% CI 212.7 to 358.3). Mean levels in the four groups were: affluent food-serving pubs (n = 16) 188.1 μg/m3 (95%CI 128.1 to 248.1); affluent non food-serving (n = 16) 186.8 μg/m3 (95%CI 118.9 to 254.3); deprived food-serving (n = 17) 399.4 μg/m3 (95%CI 177.7 to 621.2); and deprived non food-serving (n = 15) 365.7 μg/m3 (195.6 to 535.7). Levels were higher in pubs in deprived communities: mean 383.6 μg/m3 (95% CI 249.2 to 518.0) vs 187.4 μg/m3 (144.8 to 229.9); geometric mean 245.2 μg/m3 vs 151.2 μg/m3 (p = 0.03). There was little difference in particulate levels between food and non food-serving pubs.Mean PMThis study adds to the evidence that the UK government’s proposals for partial smokefree legislation in England would offer the least protection to the most heavily exposed group - bar workers and customers in non food-serving pubs in deprived areas. The results suggest these proposals would work against the UK government’s stated aim to reduce health inequalities. Second hand smoke (SHS) exposure causes of a range of serious adverse health effects in children, adults and pregnant women -3. In thChoosing Health proposed introducing partial smokefree legislation for England with exemptions including for pubs and bars that do not prepare and serve food, and private members clubs  2004 ) by mappWe aimed to perform air quality measurements in four pubs per evening, one from each category, visited in sequence. To ensure a range of measurements we sampled on four separate days of the week (Tuesday-Friday). Pub occupancy may vary by day of the week and time of night. Therefore, we included a pub from each category on every evening and varied the order of monitoring the pubs so that a pub from each of the four categories was monitored once first, once second, once third and once fourth within each of the four study areas. Occasionally, to minimise transport time, the order of visiting pubs or choice of pubs had to be slightly modified from this plan.TSI SidePak AM510 Personal Aerosol Monitor – TSI, Inc., St. Paul, USA) in a small bag. The monitor was zero calibrated each evening. It was fitted with a 2.5 μm impactor to sample and record the average levels of RSP (PM2.5) over one minute periods. The air flow rate was 1.7 l/min. A length of Tygon™ tubing was attached to the inlet of the SidePak, with the other end left protruding outside the bag. The recorded measurements were downloaded to a PC for analysis. A calibration factor of 0.32 was applied based on calibration work with a ThermoMIE personalDataRAM model pDR-1200 real-time aerosol monitor . This type of monitor had been used in a previous similar study in Delaware and has been calibrated against standard pump-and-filter gravimetric methods [Data collection was carried out using portable air quality monitors according to a protocol modified from one developed for a US study . Pubs weAt each venue we identified the busiest room of the pub where smoking was allowed, and selected a central area for monitoring, away from any open external doors or windows, kitchen areas, and not in the immediate vicinity (< 1 metre) of anyone smoking (unless this were not possible). Smoke free areas or rooms were avoided. To avoid affecting occupants' behaviour, investigators acted as normal customers and bought a drink before moving to the sampling area.The bag was carried or placed on a seat or table wherever possible to sample the ambient air. We recorded the place of monitoring within the venue and time of entry. Recording occurred for at least 30 minutes and usually lasted about 30–35 minutes. We recorded whether or not special events likely to affect occupancy, such as quiz nights, were taking place.2.5 level for each venue, and the mean PM2.5 levels by the four pub types and by deprivation and food-serving categories separately. PM2.5 levels generally had a highly positively skewed distribution, so significance tests were performed using log transformed data, and geometric means are also reported where appropriate.We calculated a time-weighted average PMEthical approval was not required for this study as no measurements were made on human subjects.Of 402 pubs in the sampling frame, 71% were in deprived areas (IMD4-5). Only nine refused to take part. Monitoring was completed in 64 pubs, 16 in each study area. Equal numbers from each pub category were sampled on each evening except for one night in Burnley when a food-serving pub from a deprived area was substituted for a non food-serving pub.The mean time of starting monitoring by type of pub was as follows: affluent food-serving 8.58 pm ; affluent non food-serving 9.17 pm (range 7.50 to 10.40 pm); deprived food-serving 8.55 pm (range 7.45 to 10.25 pm); deprived non food-serving 9.08 pm (range 7.36 to 10.20 pm); and all affluent 9.08 pm (range 7.15 to 10.50 pm) and all deprived 9.01 pm (range 7.36 to 10.25 pm).Figure 2.5 across all venues was 285.5 (95% CI 212.7 to 358.3) μg/m3. Levels were highest on Thursdays (mean PM2.5 = 356.7 μg/m3) and lowest on Tuesdays (mean PM2.5 = 187.3 μg/m3). Mean levels varied by order of visit as follows: 287.3 μg/m3 for the first, 209.5 μg/m3 for the second, 311.6 μg/m3 for the third, and 333.4 μg/m3 for the final visit.The mean level of PM2.5 levels by pub category. Figure 2.5 were higher in pubs in deprived communities  than in pubs in more affluent areas , p = 0.03. There was little difference in levels between food and non food-serving pubs .Table 2.5.The English Public Health White Paper proposed to exempt non food-serving pubs and bars from the  smokefree legislation. This study was designed to investigate air quality in pubs in relation to this proposal, and the degree to which air quality varied by deprivation. This is the first such investigation that we are aware of in the UK or elsewhere. We found that levels of fine particles were about twice as high in pubs situated in more deprived areas, but there was little difference between food and non food-serving pubs. Our measurements spanned a range of days and times, and included relatively quiet times early on in midweek evenings. We excluded Saturday nights, when occupancy and hence smoking levels are likely to be at their greatest. Despite this four pubs still had extremely high levels of PM2.5 in these hospitality venues, of the same order as those found in similar studies of 20 hospitality venues from New York (mean 324 μg/m3), [3) [Fine particle levels have been used in previous studies of air quality in the workplace, including in the hospitality trade ,12. We f μg/m3),  and eigh2.5 observed were much higher than seen in heavily trafficked urban areas. For example, mean ambient PM2.5 levels in central London (Marylebone Road) in September 2005 were 23.5 μg/m3, 24.6 μg/m3 between 7 and 12 pm. [2.5, though one may be introduced soon. In the USA, the Environmental Protection Agency (EPA) uses an air quality standard for ambient air, one component of which is the mean PM2.5 level over a 24 hour period [3 are labelled by the EPA as 'unhealthy', 'very unhealthy' and 'hazardous' respectively [3), 19 were in deprived areas. All the pubs with very high levels – two 500–999 μg/m3and four > 1000 μg/m3 – were in deprived areas.Levels of PMd 12 pm.  In the Ur period . Levels ectively . Direct 3 seen in four pubs in our sample show how poor air quality can be in the smokiest venues.A limitation of the study was the lack of information on ventilation in the participating pubs. However, the data represent current figures from real world settings and should therefore be representative of air quality experienced by customers and staff across a range of UK pubs. As noted above, the lack of sampling on Saturday nights means that we may have missed periods when air quality is poorest. The very high levels of over 1000 μg/mIn conclusion, we found high levels of fine particles in 64 pubs from across the north west of England. Air quality was poorest in pubs situated in more deprived areas. Most 86%) pubs in the sampling frame from the four study areas were in IMD deprivation quintiles 3 to 5, and 71% were in quintiles 4 or 5. The equivalent figures were 82% and 65% respectively in North West pubs from 15 Local Authority areas 6% pubs i suggest No specific funding was allocated to this study. Richard Edwards was unpaid chair of NW ASH and Claire Probert paid campaign director of NW ASH at the time of the study. All other authors have no competing interests to declare.All authors contributed to developing the original idea for the study and the study protocol. Data collection was carried out principally by CPH, KH, CP and RH with additional help from the other authors. The dataset for analysis was prepared by RE, CPH and AFRW. Analysis was carried out mainly by RE with additional suggestions from AFRW and MvT. The paper was drafted by RE. All authors contributed comments on the first and subsequent drafts, and approved the final version for submission. RE is the guarantor of the study.The pre-publication history for this paper can be accessed here:


Resistance to cytotoxics precludes the successful treatment of many solid tumours. Inhibition of DNA synthesis in normal tissues with antimetabolites such as hydroxyurea (HU) may be a useful means of improving the selective uptake of toxic thymidine analogues by the relatively resistant tumour cells. HU also inhibits DNA repair by the critical depletion of intracellular deoxyribonucleotides. Twenty-five patients with various malignancies received 5-iodo-2-deoxyuridine (IUdR) 100 mg m-2 as a 20 min i.v. infusion and the uptake of IUdR was determined 1 h later immunocytochemically. Of these patients, 14 received IUdR 23 h from the start of a continuous i.v. infusion of HU (36 g over 36 h). Uptake of IUdR was equally suppressed in bone marrow and tumour aspirates, 0.1% (+/- 0.2%) of marrow precursor cells and 0.5% (+/- 0.4%) of tumour cells respectively, in patients who received HU compared to the uptake of IUdR in 11 patients who were not given HU 6.8% (+/- 1.1%) and 12.2% (+/- 1.8%) respectively. Mean HU plasma concentrations at the time of IUdR administration was 1.7 +/- 0.2 mM. The growth fraction of tumour cells (using Ki67 labelling) was not changed after treatment with HU. It is concluded that (1) since DNA synthesis is effectively inhibited by HU in tumour cells, differential uptake of radiolabelled IUdR by those cells will not be feasible using the current schedule of HU administration, (2) HU may be used as an inhibitor of DNA repair in vivo since the degree of inhibition correlates with that required to inhibit repair experimentally and that (3) Ki67 labelling index is not useful in studying cell kinetics in patients treated with HU.


The aim of the project was to use current simple and practical laboratory tests and compare results with the foaling rates of mares inseminated with commercially produced frozen semen. In Exp. 1, semen was tested from 27 and in Exp. 2 from 23 stallions; 19 stallions participated in both experiments. The mean number of mares per stallion in both experiments was 37 . Sperm morphology was assessed and bacterial culture performed once per stallion. In Exp. 1, progressive motility after 0, 1, 2, 3, and 4 h of incubation using light microscopy, motility characteristics measured with an automatic sperm analyzer, plasma membrane integrity using carboxyfluorescein diacetate/propidium iodide (CFDA/PI) staining and light microscopy, plasma membrane integrity using PI staining and a fluorometer, plasma membrane integrity using a resazurin reduction test, and sperm concentration were evaluated. In Exp. 2, the same tests as in Exp. 1 and a hypo-osmotic swelling test (HOST) using both light microscopy and a fluorometer were performed immediately after thawing and after a 3-h incubation. Statistical analysis was done separately to all stallions and to those having ≥ 20 mares; in addition, stallions with foaling rates < 60 or ≥ 60% were compared. In Exp. 1, progressive motility for all stallions after a 2 – 4-h incubation correlated with the foaling rate (correlation coefficients 0.39 – 0.51), (p < 0.05). In stallions with > 20 mares, the artificial insemination dose showed a correlation coefficient of -0.58 (p < 0.05). In Exp. 2, the HOST immediately after thawing showed a negative correlation with foaling rate (p < 0.05). No single test was consistently reliable for predicting the fertilizing capacity of semen, since the 2 experiments yielded conflicting results, although the same stallions sometimes participated in both. This shows the difficulty of frozen semen quality control in commercially produced stallion semen, and on the other hand, the difficulty of conducting fertility trials in horses. Tischner . Th. ThTischon semen , 7] and andTischon semen . In Malms review , conflics review . TherefoAmann [Graham [Amann  stated tAmann -13,7. Gr [Graham  listed sObtaining good fertility data is difficult in horses. The number of mares and stallions used is too small, too few mares are inseminated at the appropriate time using adequate AI doses, too few semen samples are evaluated in an appropriate manner from each male, and the fertility data of mares is inaccurate .Sperm membranes are particularly vulnerable during freezing . This suThe aim of the present study was to use economically feasible and simple laboratory tests and correlate them with the foaling rates of mares. The pregnancy rates per cycle would have better reflected fertility , but theResults of frozen semen evaluation tests and foaling rates of mares were compared in 2 experiments. In the first experiment, semen of 27 stallions was tested and in the second experiment semen of 23 stallions; 19 stallions participated in both experiments. Only stallions having foaling data from at least 7 mares were included; the data were also analyzed separately for stallions having ≥ 20 mares.Semen straws, frozen between 1988 and 1997, were available from 27 commercial stallions from Sweden (22), Finland (2), Italy (2), and the USA (1). Twelve of the stallions were American Standardbreds and 15 others represented various breeds of riding horses. Semen from one stallion was frozen in 5-mL straws, from 22 in 2.5-mL straws and from 4 stallions in 0.5-mL straws. The foaling data originated from Finland and Sweden from 1989 to 1998. The mean number of mares per stallion was 37 . The average foaling rate was 56% . Twelve stallions had foaling rates > 60% and 15 had foaling rates < 60%.Semen straws, frozen between 1988 and 1998, were available from 23 commercial stallions from Sweden (18), Finland (3), Italy (1) and Germany (1). Semen from 18 stallions was frozen in 2.5-mL straws and 5 in 0.5-mL straws. Seven stallions were American Standardbreds and 16 were various breeds of riding horses. The foaling data originated from Finland and Sweden from 1989 to 1999. The average number of mares per stallion was 37 . The mean foaling rate was 60% . Fourteen stallions had foaling rates of > 60% and nine < 60%.In the first experiment, the semen evaluations were performed once immediately after thawing, but motility assessment using light microscopy was continued for 4 h. Since the incubation appeared to differentiate sperm more readily than examination immediately after thawing, all tests were carried out 0 and 3 h after thawing in the second experiment. Bacterial culture was performed only in the first experiment and the hypo-osmotic swelling test (HOST) only in the second experiment. The HOST was included in the evaluation tools due to promising results in stallions ,18. The 6 spermatozoa/mL.The 0.5-mL straws were thawed at 37°C for 30 sec, the 2.5-mL straws at 50°C for 40 and the 5-mL straws for 45 sec. The semen concentration was measured in a Bürker counting chamber, and the total number of spermatozoa per straw calculated. An insemination dose was one straw when the 2.5- or 5-mL straws were used and from 1 to 10 straws for the 0.5-mL straws. The semen was extended with a warm (+30°C) skim milk extender  to a conThe sample for the longevity test was prepared by placing 0.5 mL of extended semen into a 3-mL vial enclosed with a cap. The sample was kept in a water bath at 37°C for 4 h (Exp. 1) or 3 h (Exp. 2). The total and progressive motility and velocity were evaluated by light microscope every hour (Exp. 1) or after 3 h (Exp. 2).Varner et al. [The post-thaw motility was evaluated with a light microscope for the percentage of progressively motile spermatozoa, total motility percentage and a velocity score (from 1 to 3). Motility parameters were measured with an automatic sperm analyzer  using video taping . A 7-μL r et al. . When thPlasma membrane integrity was evaluated after thawing, using 3 methods in Exp. 1 and 5 methods in Exp. 2: 1) carboxyfluorescein diacetate/propidium iodide (CFDA/PI) staining and counting of cells with a fluorescence microscope, 2) PI staining and measurement with a fluorometer , 3) resazurin reduction test with a fluorometer, 4) HOST and counting cells with a microscope (only in Exp. 2) and 5) HOST using a fluorometer (only in Exp. 2). In Exp. 1, the tests were performed once immediately after thawing, while in Exp. 2 they were repeated after a 3-h incubation.6 spermatozoa/mL. Aliquots of 20 μL of CFDA stock solution consisting of 0.46 mg CFDA in 1 mL of DMSO (dimethylsulpfoxide) and 10 μl of PI stock solution (0.5 mg PI in 1 mL of 0.9% NaCl solution) were taken, mixed with 950 μl of semen, and incubated for 8 min at 30°C [For evaluation of plasma membrane integrity with CFDA/PI staining, the semen was extended with a skim milk extender  to a con at 30°C . A 5-μL 6 spermatozoa/mL) and PI solution were dispensed into a well and shaken gently for 2 min. Spermatozoa from the same samples were killed by unprotected rapid freezing and slow thawing to obtain internal control samples consisting of only non- viable cells (100% fluorescence). The control sample was immersed in liquid nitrogen for 1 min and thereafter allowed to stand at room temperature for 30 sec and then 3 min in a water bath (37°C). Blanks containing 50 μl of diluted extender and 50 μl of PI were analyzed separately for every experiment in 4 replicates; the incubation time was 8 min. The percentage of fluorescence was calculated from the ratio of fluorescence intensities of the rapidly frozen control sample and the sample to be analyzed, after comparing with the blank values [The second plasma membrane viability test was performed using an automatic fluorometer , which reads a 96-well microtitration tray and has an incubation compartment. The interference filter at the excitation path and that of the emission filter showed maximum transmission at 544 nm and 590 nm, respectively. For the fluorometric assay, 20 mg of PI was dissolved in 1 L of Beltsville Thawing Solution (BTS)  and dispensed in 3-mL aliquots. Equal aliquots (50 μL) of BTS diluted semen sample . An aliquot of 0.125 mL of sperm was added to 0.5 mL of solution and the mixture was incubated for 30–45 min at 37°C. A 5-μL drop was placed on a slide and overlaid with a cover slip. A total of 200 spermatozoa per sample were evaluated for the presence of bent tails in light microscopy [6 spermatozoa/mL). The fluorometric method was the same as for PI-stained semen. The mixture was incubated at 37°C and analyzed again after 3 h.For the HOST, semen was extended to 4 × 10croscopy  and alsoWatson [Blom [The frozen-thawed semen smears were air-dried and stained with Giemsa according to Watson . A totalon [Blom .The bacterial culture was performed by spreading a drop of each sample onto half a blood agar plate, using a 10-μL sterile loop. After incubation for 24 and 48 h at 37°C, colony forming units (CFUs) were counted and bacterial species recognized. If more than 100 CFUs were detected per sample, the number was not calculated further.Pearson and Spearman correlation coefficients were used to study the association between the parameters. The results were accounted for, if both correlation coefficients were congruent. P-values < 0.05 were considered significant. The results were expressed as mean ± the standard error of the mean (s.e.m.). The stallions were divided into 2 groups: foaling rate of mares < 60% or > 60%. The independent sample t-test was used to test differences in the laboratory test parameters between the 2 groups of stallions. Statistical analysis was also performed separately from the material restricted to those stallions having > 20 mares .The percentage of normal spermatozoa varied from 51% to 89%. Major abnormalities accounted for 9.5%, including head abnormalities in 4.1% (1–12%), tail bent under the head 2.5% (0–6%), and mid piece defects 2.4% (0–7%); minor abnormalities comprised 10.7% (3–31%) including mainly bent tails 6.9% (1–29%), normal loose heads 1.4% (0–7%), and loose acrosomes 2% (0–5%). There was no association between morphological findings and foaling rate.Enterococcus, Enterobacteriaceae, or Corynebacteriaceae. There was no association between bacteriological findings and foaling rate.A total of 52% of the samples showed no microbial growth, in 41% < 100 CFUs per plate were detected, and in 7% > 100 CFUs per plate. The microbes were mainly coagulase-negative staphylococci or belonged to the families Average CASA motility and s.e.m. were as follows: TMOT 37.0 ± 3.3, PROG 27.6 ± 2.7, and VAP 58.7 ± 2.0; these values did not correlate with fertility. Average progressive motility evaluated in light microscopy showed following changes during incubation: 0 h 40.2 ± 1.7 , 1 h 35.0 ± 1.4 , 2 h 29.0 ± 1.6 , 3 h 24.9 ± 1.7 (10–40), and 4 h 21.0 ± 1.7 (5–40). Progressive motility correlated significantly with foaling rate after 2–4 h of incubation . Stallions (> 7 mares) with foaling rates of > 60% appeared to retain sperm motility slightly better than stallions with foaling rates of < 60%, although the difference was not statistically significant . The total number of sperm/AI dose and sperm concentration for stallion groups having foaling rates < 60% or > 60% are shown in Fig. When the various parameters were compared with each other, all motility parameters correlated significantly with each other (correlation coefficients varied from 0.44 to 0.81), similarly the plasma membrane integrity tests showed significant correlations between each other (0.37 – 0.83). CFDA/PI staining with light microscopy and with a fluorometer correlated significantly also with progressive motility before incubation. The total number of sperm/AI dose showed a significant negative correlation with the other parameters, except with progressive motility during incubation (3–4 h) and CFDA/PI with light microscopy.The average HOST-values were 30.1 ± 1.6 18–49) before incubation and 21.7 ± 1.6 (9–46) after 3 h of incubation. A significant correlation coefficient of -0.50 with foaling rate (p < 0.05) was demonstrated before incubation. The average CFDA-values obtained in microscopy were 42.9 ± 2.4 (14–66) before incubation and 33.0 ± 1.8 (12–48) after the 3-h incubation. When stallions having > 20 mares were analyzed, CFDA/PI staining with light microscopy at 0-h incubation and HOST with fluorometer after a 3-h incubation showed correlation coefficients of 0.5 with foaling rate (p > 0.05). The HOST results in 2 stallion groups divided by their foaling rates are shown in Fig. 9 before When the various parameters were compared, TMOT, PROG, VAP, and RAP correlated after the 0-h and 3-h incubations, correlation coefficients ranging from 0.5 to 0.8. CFDA, HOST and resazurin both by microscopy and fluorometer correlated after the 0-h and 3-h incubations with coefficients of 0.4 – 0.8, but no correlation was demonstrated between these parameters and parameters depicting motility. Before incubation, the concentration showed a significant negative correlation with CFDA/PI staining, using both light microscopy and the fluorometer, but this correlation disappeared after the 3-h incubation.No single test was found to be a consistently reliable method for predicting the fertilizing capacity of semen. The results of Exp. 1 could not be repeated, although the stallions and methods used were partly similar. Differences in the outcome of these experiments could be explained also by different ejaculate batches.Some tendency for the plasma membrane integrity tests to be indicators of higher foaling rates was noted in our study. Fluorometers have not been much used in the examination of stallion semen. Although no strong correlation with foaling rate was found, the various fluorometric measurements did correlate with plasma membrane integrity in light microscopy and with each other. The use of fluorometers has some advantages in comparison to the use of light microscopy: it requires less time, and more cells in the sample are assessed . In boarNeild et al. [Nie et al. [Blach et al. [Samper [d et al.  found noe et al.  reportedh et al.  found in [Samper  who noteVoss and his coworkers [Jasko et al. [Newcombe [Kirk et al. [Voss et al. [Motility correlated with fertility in our first experiment but not in the second. One explanation can be pre-selection of semen. Most laboratories use 30% as a cut-off value when accepting semen for sale. The most important task of semen tests is to exclude semen of inferior quality, and this had already been done in the laboratories which had exported the semen. Another explanation is the use of different batches from several ejaculates from each stallion, but still the question on the accuracy of motility evaluation as a means of predicting fertility is raised. Semen from a stallion with the highest progressive motility 60%) did not produce any pregnancies in 10 inseminated mares. Motility has been claimed by other investigators to be a poor predictor of pregnancy rates 0% did no. Howevero et al.  reportedNewcombe  reportedk et al. . The motAmann [6 frozen sperm [The total number of sperm/AI dose and concentration correlated negatively with many parameters. This can be explained by the tendency to increase AI dose and thus the concentration – the volume of one straw is limited – when low post-thaw motility is detected to increase the number of progressively motile spermatozoa and consequently the possibility of pregnancy . This prAmann  presenteAmann ,37. Speren sperm .Jasko et al. [Voss et al. (1981) noted considerable variation in the morphologic characteristics among stallions and among ejaculates within stallions. They suggested that spermatozoal morphology may not be as valuable in evaluation of potential fertility in the stallion as it is in other large domestic animals.In our study, the percentage of morphologically normal semen and foaling rates did not correlate, perhaps because semen with a high percentage of morphological abnormalities is not frozen. o et al. ,32 showeSamper et al. [Problems confronted in previous fertility studies are also obvious here. The number of mares inseminated per stallion was small, the straws were not from the same batch, foaling rates were collected during several years – fertility of stallions can vary between years and decrease with age. Insemination conditions, veterinary skills, management of stud farms, criteria for mare selection, etc. vary considerably. Many other factors in addition to semen characteristics influence fertility. Although obtaining pregnancy rates is difficult, they are more accurate and reliable than foaling rates. However, this does not eliminate other factors affecting fertility, such as management and reproductive performance of mares, their previous reproductive status, and possible genetic factors . A multir et al.  summarizNie et al. [Development of freezing methods for stallion semen is dependant on finding dependable correlations between laboratory tests and fertility, which appears very difficult to achieve since the results of different studies in this field tend to be contradictive. The present study was unable to address the question of which laboratory tests would accurately predict fertility of commercially produced stallion semen. Objectivity, repeatability, and accuracy are basic requirements for laboratory assays, but many semen analysis tests do not meet these requirements . Qualitye et al.  concludeThe constraints in horse breeding – small numbers of mares per ejaculate and per stallion and the tremendous variations in mare management and insemination – never allow us to carry out trials similar to what the cattle industry has done in developing freezing methods and AI techniques. Fertility trials of horses are bound to be of little value because of these reasons .


One hundred and fifty-nine previously untreated patients with small cell lung cancer (SCLC), who were not eligible for intensive chemotherapy, were entered into a randomised study of intravenous (i.v.) doxorubicin and ifosfamide (with mesna) and oral etoposide. The i.v. drugs were given either by bolus therapy or by a continuous infusion (CI) pump over 7 days via a central venous line. Therapy was given for 6 weeks only. On weeks 1, 3 and 5 IV doxorubicin 35 mg m-2 was given with 5 days of oral etoposide 100 mg m-2 daily. On weeks 2, 4 and 6 IV ifosfamide 5 g m-2 was given with equidose mesna. The overall median survival was 25 weeks for patients in the bolus arm and 30 weeks for the CI therapy (P = 0.45). The overall response rate was 64% (18% complete response-CR) and 69% (30% CR) respectively (P = 0.13). The median WHO score for haematological toxicity was 4 for bolus therapy and 3 for CI therapy (P = 0.0007). Despite a trend for less supportive care for patients on CI therapy there were no significant differences in the use of i.v. antibodies and blood or platelet transfusions. There were fewer treatment delays due to myelotoxicity in the CI arm (P = 0.04). The median WHO score for non-haematological toxicity was 2 in both treatment groups. There was significantly less nausea (P = 0.037) but more mucositis (P = 0.01) in the CI arm. Weekly chemotherapy using CI treatment was as effective as bolus therapy. It was well accepted by patients. The assessment of quality of life in a subgroup of patients showed a statistically significant reduction in anxiety and depression for both groups of patients during therapy.


CYP1A2*1F and COMT Val58 Met polymorphisms among 332 breast cancer cases and 254 controls in the Hawaii component of the Multiethnic Cohort.Mammographic density is a strong predictor of breast cancer risk and is increased by hormone replacement therapy (HRT). Some associations with genetic polymorphisms in enzymes involved in estrogen metabolism have been described. This cross-sectional analysis examined the relation between mammographic density and the Mammographic density, before diagnosis in cases, was quantified by using a validated computer-assisted method. Blood samples were genotyped by standard PCR/RFLP methods. Adjusted mean percent density was calculated by genotype using mixed models with the unstructured covariance option.CYP1A2*1F gene and percent density, but not the dense area, was suggested (p = 0.11). The relation was limited to controls (p = 0.045), postmenopausal women not using HRT (p = 0.08), and normal weight subjects (p = 0.046). We did not observe any relation between the COMT Val58 Met polymorphism and breast density.A positive association between the C allele in the CYP1A2 genotype and the size of the dense areas suggests an effect on the non-dense, i.e., fatty breast tissue. The discrepancies among studies may be due to differential susceptibility; changes in enzyme activity as a result of the CYP1A2*1F polymorphism may influence breast tissue differently depending on hormonal status. Larger studies with the ability to look at interactions would be useful to elucidate the influence of genetic variation in CYP1A2 and COMT on the risk of developing breast cancer.The lack of an association between the  CYP1A2*1F gene and the Met allele in the COMT gene [CYP1A2*1F was also significantly associated with lower serum estradiol levels during the luteal phase [CYP1A2*1F gene had a lower risk of breast cancer than women with the common alleles [COMT gene, higher breast density was found for postmenopausal HRT users in one report [CYP1A2*1F and the COMT Val58 Met polymorphisms among the overlapping subjects.Mammographic density has been shown to be independently associated with breast cancer risk . AlthougOMT gene . The CC al phase . The paral phase ,17. Alsoal phase . In a ca alleles , but a c alleles . As for e report , lower be report , lower be report , higher e report , and no e report . To clare report  with a me report  and examThe subjects for this analysis were originally recruited for two separate case-control studies ,20 nesteTo assess breast density, the cranio-caudal views for both sides of several mammographic examinations were retrieved . All mamCOMT Val58 Met and CYP1A2*1F (A-154C) polymorphisms as described previously [DNA was extracted from white blood cells and genotyped by PCR/RFLP methods for the eviously .All statistical analyses were performed using SAS version 9  . In ordeDue to the study design, nearly half of the 575 subjects were breast cancer cases Table . On averCYP1A2*1F polymorphism among all women; the difference between the AA and CC genotypes was 5.1% (p for trend = 0.11) . Although we detected no significant interaction effects between the CYP1A2*1F polymorphism and case status, menopausal status, and BMI , we noted a number of differences after stratification by these variables. The association between the CYP1A2*1F allele and percent density was stronger among controls than cases. The difference between subjects with AA and CC genotype was 8.9% (p = 0.045), while there was no significant difference in breast density by genotype among cases (p = 0.86). Given the small number of mammograms among premenopausal women, the 9.6% difference between the two homozygous groups was not significant (p = 0.38). Among postmenopausal women, the 9.1% difference for mammograms taken without HRT use was close to significance (p = 0.08), whereas the difference among HRT users was only 2.5% (p = 0.48). Similarly, the positive association was limited to women with a BMI of less than 25 kg/m2 , whereas percent density did not vary by genotype among overweight and obese women. Stratification by ethnicity showed non-significant higher mean densities for Japanese CC carriers but no relation among Caucasians. The mean densities for the Japanese AA, AC, and CC carriers were 32.9%, 31.7%, and 39.4% (p = 0.44); the respective values for Caucasians were 26.0%, 27.9%, and 25.1% (p = 0.60).Adjusted percent densities were significantly higher with an increasing number of variant alleles in the COMT Val58 Met were not associated with percent density in the entire population or in any subgroup .Genotypes for up Table . The denCYP1A2 and COMT (p = 0.85 and 0.98).When we used the size of the dense areas as dependent variable, we found no difference in mean densities by genotype for CYP1A2 gene were 0.86 (95% CI = 0.60–1.23) and 0.62 (95% CI = 0.30–1.30), respectively. For the COMT Val58 Met polymorphism, the OR was 0.93 (95% CI = 0.64–1.35) for one Met allele and 0.61 (95% CI = 0.37–1.02) for two Met alleles as compared to the women with two Val alleles.Compared to women with the AA genotype, the risk estimates for breast cancer associated with one or two C alleles in the CYP1A2*1F gene, but the relation was limited to controls, postmenopausal women not using HRT, and normal weight women. The fact that the size of the dense areas was not associated with the CYP1A2 genotype suggests that the relation may be due to an effect on the non-dense, i.e., the fatty breast tissue. We did not observe any relation between percent density and the Met polymorphism in the COMT gene.In this analysis of longitudinal mammographic data, we observed a weak association between percent density and the number of C alleles in the p for trend = 0.01) among postmenopausal women not using HRT [CYP1A2*1F [p for trend = 0.03) that also included Latina and African-American women in Los Angeles. The findings with regard to the COMT Val58 Met polymorphism are also inconsistent. Studies in Hawaii [In comparison, a Canadian study reported mean percent densities of 14% for the lowest and 31% for the highest quartile of CYP1A2 activity  declare that they have no competing interests.YT performed the statistical analysis and drafted the manuscript. GM designed the study and helped to perform the statistical analysis and draft the manuscript. LM designed the study, supervised the genetic analysis and critically commented on the drafted manuscript. GM and LM obtained funding for the previous projects. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The risk of developing infection in diabetic patients is higher and urinary tract is the most common site for infection. Serious complications of urinary infection occur more commonly in diabetic patients. To study the prevalence and associates of asymptomatic bacteriuria (ASB) in women with type 2 diabetes mellitus in the Iranian population, this study was conducted.5 colony-forming units/ml of 1 or 2 bacterial species, in two separated cultures of clean-voided midstream urine. All the participants were free from any symptoms of urinary tract infection (UTI). Associates for developing bacteriuria was assessed and compared in participants with and without bacteriuria.Between February 10, 2004 and October 15, 2004; 202 nonpregnant diabetic (type 2) women (range: 31 to 78 years old) with no abnormalities of the urinary tract system were included in this clinic based study. We defined ASB as the presence of at least 101c level (P < 0.75), and the presence of ASB.In this study, the prevalence of ASB was 10.9% among diabetic women. E. coli was the most prevalent microorganism responsible for positive urine culture. Most of the isolated microorganisms were resistant to Co-trimoxazole, Nalidixic acid and Ciprofloxacin. Pyuria (P < 0.001) and glucosuria (P < 0.05) had a meaningful relationship with bacteriuria but no association was evident between age (P < 0.45), duration of diabetes (P < 0.09), macroalbuminuria (P < 0.10) and HbAThe prevalence of ASB is higher in women with type 2 diabetes, for which pyuria and glucosuria can be considered as associates. Routine urine culture can be recommended for diabetic women even when there is no urinary symptom. Diabetes leads to several abnormalities of the host defense system, and higher glucose concentration in urine may serve as a culture medium for pathogenic microorganisms as well. The risk of developing infection in diabetic patients is higher ,2 and ur1c were obtained while patients were fasting. Urine samples were checked for Macroalbuminuria by dip stick. Mid-stream clean voiding urinary specimens were collected for urinalysis, microscopy, culture and sensitivity. The specimens were refrigerated immediately and cultured within two hours. All urine samples were cultured on Blood and MacConkey agar plates. The plates were incubated at 37°C aerobically for 48 hours.This was a cross sectional, descriptive and analytic study. Participants were recruited between February 10, 2004 and October 15, 2004 from type 2 diabetic women who had referred to Tehran Heart Center OPD (Out Patient Department) clinics. Patients were asked by their treating physicians to participate voluntarily with a response rate of 92%. Inclusion criteria considered as women with type 2 diabetes mellitus. Exclusion criteria was pregnancy, recent hospitalization or surgery (< 4 month), known urinary tract abnormalities (including cystopathy or recent urinary tract instrumentation), symptoms of UTI  or the use of antimicrobial drugs in the last 14 days. All patients were interviewed and their medical histories were obtained using a standardized questionnaire. Also, laboratory values such as blood urea nitrogen, serum creatinin, glucose and glycosylated hemoglobin A5 colony forming units/ml of 1 or 2 bacterial species in a culture of clean-voided midstream urine confirmed by a second culture. Presence of at least three different microorganisms in a urine specimen was considered as contamination.Bacteriuria was defined as the presence of at least 10According to the 1998 WHO criteria, diabetes mellitus was defined as fasting glucose concentration of at least 6.1 m mol/ l (110 mg/dl) or a two-hour post prandial glucose concentration of at least 10.0 m mol (180 mg/dl) or the use of glucose-lowering medication (tablets or insulin). Type 2 Sample size of this study was calculated based onwhich was equal to 200.1c, BUN and creatinine). For nominal variables we used chi squared and Fisher exact test. Mann-Whitney U test was used for dichotomous and ordinal variables . Data were analyzed  by SPSS statistical software and P value of < 0.05 was considered significant. Mean values are reported as mean ± standard deviation.Differences between patients with and without ASB were obtained through t test for continuous variables  (p < 0.001). 45.5% of patients with bacteriuria also displayed pyuria and 86.5% of participants without bacteriuria showed no evidence of pyuria.We observed a significant relationship between bacteriuria and glucosuria (P = 0.03). Among patients with bacteriuria, 59.1% had no glucosuria but 9.1%, 9.1% and 22.7% of them had trace, +1 and +2 glucosuria respectively.Among participants without bacteriuria, 80.8% displayed no glucosuria. However, 4.5%, 8.5% and 6.2% had trace, +1 and +2 glucosuria respectively.No significant association was found between bacteriuria and the age of the participants (p = 0.45). Among patients with bacteriuria, none was under 40 years old. Of patients between 40-49, 50-59 and more than 60 years, 19.2%, 11.2% and 9.2% had bacteriuria respectively.No evidence of a significant relation between bacteriuria and the duration of diabetes (p = 0.09) was found. Among patients with less than 10 years of diabetes, 10.1% (n = 15), and patients with 10-20 years of diabetic history, 20.6% (n = 7) had bacteriuria. But among the 13 patients who had diabetes of more than 20 years duration, none had bacteriuria. No significant relationship was found (p = 0.10) in regard to the relationship between bacteriuria and macroalbuminuria. Of the 22 patients who had positive urine culture, 77.3% (n = 17) did not have macroalbuminuria. Among 24 participants with macroalbuminuria, 20.8% (n = 5) had bacteriuria as well.1c levels (p = 0.75). 57.1 % (n = 12) of the patients with HbA1c levels of less than 8 had bacteriuria, and 42.9% (n = 9) of the patients whose HbA1c levels were 8 or more, had also developed bacteriuria.In this study, no significant association was evident between bacteriuria and HbAIn this study 22 out of 202 (10.9%) type 2 diabetic women had ASB. This is comparable with studies by Kayima et al  in 1996 As most other previous studies, E. coli was the most prevalent microorganism (59.1%) isolated from urine cultures of our participants. In a few studies, the microorganism was different. For example Kelebsiella was the most common organism in the Alebiosu study .As table According to our statistical analysis and with regard to the calculated P values, it seems that pyuria (p < 0.001) and glucosuria (p < 0.03) have a significant relationship with bacteriuria. So, pyuria and glucosuria can be associated with ASB in diabetic women.Even though age is a well-known risk factor for bacteriuria in women without diabetes  and someSome studies have shown that a longer duration of diabetes increases the risk of developing ASB ,13, whilSome studies found macro albuminuria as a risk for developing ASB ,21, howe1c is not an associate for ASB.In confirmation of other studies ,21, our Finally, we suggest a periodical urine culture be taken from diabetic patients older than 40 years to detect ASB early on (and possibly prevent its development or persistence). Further studies need to be done to assess the prevalence of asymptomatic bacteriuria in diabetic women in other Iranian population and also to assess their antimicrobial sensitivity pattern. Long-term follow up should also be implemented for diabetic patients with ASB so that the precise conclusions could be drawn regarding the course of UTI development in the future.The prevalence of ASB is higher in women with type 2 diabetes, for which pyuria and glucosuria can be considered as associates. Routine urine culture can be recommended for diabetic women even when there is no urinary symptom.The author(s) declare that they have no competing interests.MAB conceived of the study, contributed in analysis and interpretation of data, reviewed the manuscript and gave final approval. LS assisted in study conception & design and involved in coordinating the research and sample collection. SHA supervised data entry, carried out the analysis and drafted the manuscript. MS contributed to study design & conception, assisted with data processing and reviewed the manuscript. EK assisted in the design of the survey, assisted with data processing and contributed in interpretation of data. SF involved in planning, coordinating the research and helped in drafting the article. All the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: