We investigated these factors in the fibrotic response to bleomycin of p47Mice are administered by intranasal instillation of 0.1 mg bleomycin. Either 24 h or 14 days after, mice were anesthetized and underwent either bronchoalveolar lavage (BAL) or lung removal.phox -/- mice. At day 1, the bleomycin-induced acute inflammatory response (increased neutrophil count and MMP-9 activity in the BAL fluid) was strikingly greater in KO than wild-type (WT) mice, while IL-6 levels increased significantly more in the latter. Hydroxyproline assays in the lung tissue 14 days after bleomycin administration revealed the absence of collagen deposition in the lungs of the KO mice, which had significantly lower hydroxyproline levels than the WT mice. The MMP-9/TIMP-1 ratio did not change at day 1 after bleomycin administration in WT mice, but increased significantly in the KO mice. By day 14, the ratio fell significantly from baseline in both strains, but more in the WT than KO strains.BAL cells from bleomycin treated WT mice showed enhanced ROS production after PMA stimulation, whereas no change was observed with BAL cells from p47These results suggest that NADPH-oxidase-derived ROS are essential to the development of pulmonary fibrosis. The absence of collagen deposition in KO mice seems to be associated with an elevated MMP-9/TIMP-1 ratio in the lungs. This finding highlights the importance of metalloproteinases and protease/anti-protease imbalances in pulmonary fibrosis. Pulmonary fibrosis is a severe chronic disease with various causes and poor prognosis. Its main histological features include lesions of the alveolar septa, fibroblast and myofibroblast proliferation in lung parenchyma, abnormal reepithelialization, and excessive extracellular matrix macromolecule deposition -3. Lung Metalloproteinases (MMPs) and their specific inhibitors, the tissue inhibitors of MMPs (TIMPs), are the hallmark of this fibrogenic microenvironment. MMPs are key enzymes that regulate tissue remodeling through turnover of the extracellular matrix in both normal and pathological conditions (for review see ). They pStrong evidence indicates that various MMP/TIMP imbalances are crucial elements in the fibrogenic process. Several authors suggest that a "nondegrading microenvironment" induces fibrogenicity, that is, more specifically, that various events cause TIMP-1 levels to rise in lung tissue, which in turn lowers MMP/TIMP ratios ,11,12. Bphox subunit of NADPH-oxidase [Further study is needed to illuminate the pathway that leads from lung injury, associated with ROS and acute inflammation, to initiation of the fibrogenic process, which involves remodeling mediators such as MMPs and TIMPs. The aim of the present study was to investigate the involvement of the ROS released by inflammatory cells during the development of pulmonary fibrosis and to consider the consequence on MMP/TIMP balances. We therefore examined the fibrogenic response to bleomycin administration in mice deficient for the p47-oxidase  and anal®, 2 mg/mL) from Janssen-Cilag ; acrylamide, sodium dodecyl sulfate (SDS), Tris, and BSA from Eurobio ; ELISA kits for IL-6, TIMP-1, and pro-MMP-9 detection from R&D Systems ; formaldehyde from Merck ; isopentane from Prolabo ; a low-range weight marker for SDS-PAGE from Biorad ; and an ABEL® chemiluminescence kit for measurement of in vitro ROS release from Knight Scientific Limited .This study used the following materials, from the manufacturers mentioned: bleomycin sulfate from Bellon Laboratories ; gelatin, Triton X-100, Coomassie Brilliant Blue, EDTA, Tween 20 solution, hydroxyproline, and trypan blue from Sigma ; May-Grünwald and Giemsa stains from RAL ; sodium pentobarbital from Sanofi Santé Animale ; etomidate (Hypnomidatephox +/+ "wild-type" (WT) and p47phox -/- "knockout" (KO) mice  with C57BL/6J backgrounds [Ten week-old p47kgrounds  were houPulmonary fibrosis was induced by intranasal (i.n.) instillation  of 0.1 mg bleomycin sulfate in saline solution (50 μL/mouse). Control mice received saline vehicle only. Either 24 h or 14 days after i.n. administration, mice were quickly anesthetized by an i.p. injection of sodium pentobarbital (60 mg/kg) and underwent either bronchoalveolar lavage (BAL) or lung removal. These samples were stored at -80°C until either hydroxyproline measurement or homogenization for zymography.® . Red blood cells were eliminated by adding 3 mL of distilled water for 30 seconds and then 1 mL of KCl 0.6 M onto the pellets. After centrifugation , supernatant was eliminated and the cells were suspended in 1 mL of PBS. They were then cytospun at 700 rpm for 10 minutes  and stained with the May-Grünwald Giemsa method. Differential cell counts of 200 cells used standard morphological criteria.Mice were anesthetized with an i.p. administration (20 mL/kg) of sodium pentobarbital 0.6%. The BAL protocol called for washing the airways 10 times with 0.5 mL of 0.9% NaCl solution at 37°C with a 1 mL syringe. The BAL fluid was centrifuged , and the supernatant of the first two fractions (1 mL) divided into aliquots and frozen at -80°C until analysis. The cell pellets were then pooled with the last fractions. Total cells were counted with a Coulter Z2® detection kit.After in vitro stimulation with phorbol 12-myristate 13-acetate (PMA), 0.8 μM, ROS production was assayed with a chemiluminescence technique that used the ABELAt day 14, following BAL processing, lungs were removed and homogenized with an adapted grinder . Lung tissue homogenates were then stored at -80°C until analysis.2, 2 μM ZnCl2, pH = 8. The gels were stained with Coomassie Brilliant Blue in a solution of 25% ethanol-10% acetic acid in water and rinsed in an identical solution. Gelatinase activity appeared as clear bands against blue background. We used recombinant protein molecular weight markers (20 kDa-112 kDa) to estimate the molecular weights of the gelatinolytic bands. Relative enzyme amounts were quantified by measuring the intensity of the bands with a densitometric analyzer . Results were expressed as a percentage of the band of migration of one control BAL sample loaded onto each gel. This sample was used as an internal standard to allow further comparisons between gels.Since MMPs can degrade gelatin, zymographic techniques were used to detect MMPs in both BAL (day 1 and day 14) and lung homogenates (day 14). In nonreducing conditions and in the presence of SDS, as previously described , aliquotThe amounts of total IL-6, TIMP-1, and pro-MMP-9 were determined with ELISA methods, performed according to the manufacturer's recommendations. Assay sensitivity was 31 pg/mL for TIMP-1, 15 pg/mL for IL-6, and 8 pg/mL for pro-MMP-9.Lung tissue was lyophilized, weighed, ground to a fine powder with a mortar and pestle, homogenized in PBS pH = 7.4, and divided into aliquots. After hydrolysis for 45 min in NaOH 2 N, the hydroxyproline content was assayed in duplicate aliquots, as previously described .Results were expressed as means ± SEM. The differences between the groups for treatment and strain effects were analyzed with a nonparametric Mann-Whitney U test. Correlations between the BAL analysis data and the MMP levels and activity were assessed with the nonparametric Spearman correlation test. For each analysis, P values less than 0.05 were considered to be statistically significant.in vitro with PMA  mice.Zymography identified the following gelatinolytic bands as MMP activity: pro-MMP-9 (105 kDa), MMP-9 (86 kDa), pro-MMP-2 (70 kDa), and MMP-2 (64 kDa). At day one, pro-MMP-9 activity was significantly higher in the BAL of bleomycin-treated KO mice than in that of their bleomycin-treated WT counterparts, which in turn was significantly higher than in the control mouse BAL figure . At day At one day and 14 days after bleomycin administration, MMP-2 activity was observed in both its latent (70 kDa) and active (64 kDa) forms figure . AlthougMMP activity was also evaluated at day 14 in lung homogenates figure . HomogenWe also evaluated pro-MMP-9 as well as TIMP-1 with ELISA table . The resTable phox subunit of the NADPH oxidase complex do not develop pulmonary fibrosis after intranasal administration of bleomycin. It also suggests that an imbalance of the molar MMP-9/TIMP-1 ratio may influence the fibrogenic process in this model.This study shows that mice deficient in the p47phox-/- KO mice. Unlike antioxidant compounds, which nonspecifically target all ROS sources in the tissue, knocking out the p47phox subunit of the NADPH oxidase complex shuts down only the main pathway of phagocytic ROS production. In this study, in vitro PMA stimulation of BAL cells from KO mice produced no detectable ROS, while BAL cells from WT mice did produce ROS, as previous studies of circulating neutrophils from these mice have shown [Several studies previously reported that antioxidant treatment attenuates the bleomycin-induced oxidative burden and subsequent pulmonary fibrosis ,8,17. Move shown .Hydroxyproline content did not increase in the lungs of these KO mice. This finding reveals the absence of fibrosis and thus provides strong evidence that phagocytic ROS production is an important component of the fibrogenic environment.in vivo previously described in such KO mice [Although no ROS production was detected in KO mice, their BAL gave evidence of an acute inflammatory response, as did that from WT mice: bleomycin administration elicited acute inflammation, characterized by an influx of neutrophils and associated with increased pro-MMP-9 activity. Moreover, the response of the KO mice to the bleomycin resembled the abnormal "exuberant" inflammation  KO mice ,20,21. I KO mice .phox-/- KO mice [This acute inflammation was accompanied by significantly elevated IL-6 levels in the BAL fluids of both strains of mice on the day after bleomycin administration, levels significantly higher in WT than KO mice. Given that IL-6 is secreted primarily by mononuclear cells, specifically macrophages, the difference between strains suggests that these cells are activated more weakly in the KO than in the WT mice. Moreover, in SP-D -/- alveolar macrophages, the NADPH oxidase inhibitor apocynin inactivates NF-kappa B, the transcription factor that regulates numerous proinflammatory responses, including IL-6 release . Similar KO mice . This woMMP induction, assessed by gelatinase release, has been reported in various cases of pulmonary fibrosis in human and experimental models ,25,26. WIt is thus difficult to reach a definitive conclusion at this time about the exact function of MMPs. They play a role in promoting tissue remodeling and counterbalancing excessive matrix deposition, but may also facilitate tissue damage and disruption. Their involvement in bleomycin-induced pulmonary fibrosis has been demonstrated with the inhibition of collagen deposition by bastimastat, a nonselective MMP-inhibitor . CompletIncreased TIMP-1 expression has been observed in lung extracts and in BAL fluids after bleomycin administration and after the transfer of the active TGF-beta gene to "fibrosis-prone" C57BL/6 mice ,14. In hInterestingly, in our study, the pro-MMP-9/TIMP-1 ratio was significantly higher for KO than WT mice, at both day one and day 14. At day 1 this was due to the lower MMP-9 level and higher TIMP-1 level in the BAL from WT mice, and at day 14, only to the latter. The correlation of these levels with differences in hydroxyproline levels in the lungs of bleomycin-treated mice strongly suggests that a reduced molar pro-MMP-9/TIMP-1 ratio in BAL fluid is associated with collagen deposition, beginning as early as the inflammatory events at day 1 after bleomycin administration.phox-/- KO mice could provide great amount of other kind of protease, such as serine proteases. Indeed, neutrophil elastase was shown to have an impact on the severity of bleomycin-induced pulmonary fibrosis [The usefulness of the pro-MMP-9/TIMP-1 ratio as a marker of fibrosis nonetheless requires discussion. Although a molar ratio appears to play a protective role against fibrotic changes, MMP-9 is considered primarily to be an inflammatory mediator released by leukocytes during acute inflammatory events to facilitate their progression across the basement membrane . Moreovefibrosis ,35phox-/- KO mice to produce large quantities of ROS via the NADPH oxidase pathway inhibits the development of bleomycin-induced pulmonary fibrosis. This inhibition is associated with changes in IL-6 production and in the molar MMP-9/TIMP-1 ratio, both probably key factors in airway remodeling and fibrosis. These rapidity of these differences after bleomycin administration suggests that early inflammatory events and remodeling events may establish a favorable environment for further chronic fibrogenic processes.In summary, this study demonstrates that the inability of phagocytes from p47BAL: Bronchoalveolar lavageIL: interleukinKO: knock outMMP: matrix metalloproteinaseTIMP: tissue inhibitor of metalloproteinaseROS: reactive oxygen speciesWT: wild typeBM, SN, OL, IG:and EB have made substantial contributions to acquisition and analysis of dataBM, SN, EB and VL have made substantial contributions to conception and designBM, EB and VL have been involved in drafting the articleJMP and CPB have been involved in revising it critically for important intellectual content


Rhus verniciflua Stokes. It has long been used as a food additive in Korea and as an herbal medicine throughout Asia. Recently, butein has been shown to suppress the functions of fibroblasts. Because fibroblasts are believed to play an important role in promoting the growth of breast cancer cells, we investigated the ability of butein to inhibit the clonogenic growth of small numbers of breast cancer cells co-cultured with fibroblasts in vitro.Butein , a plant polyphenol, is a major biologically active component of the stems of We first measured the clonogenic growth of small numbers of the UACC-812 human breast cancer cell line co-cultured on monolayers of serum-activated, human fibroblasts in the presence of butein (2 μg/mL) or various other modulators of fibroblast function . In a subsequent experiment, we measured the dose-response effect on the clonogenic growth of UACC-812 breast cancer cells by pre-incubating the fibroblasts with varying concentrations of butein (10 μg/ml-1.25 μg/mL). Finally, we measured the clonogenic growth of primary breast cancer cells obtained from 5 clinical specimens with normal fibroblasts and with fibroblasts that had been pre-treated with a fixed dose of butein (2.5 μg/mL).Of the five modulators of fibroblast function that we tested, butein was by far the most potent inhibitor of clonogenic growth of UACC-812 breast cancer cells co-cultured with fibroblasts. Pre-treatment of fibroblasts with concentrations of butein as low as 2.5 μg/mL nearly abolished subsequent clonogenic growth of UACC-812 breast cancer cells co-cultured with the fibroblasts. A similar dose of butein had no effect on the clonogenic growth of breast cancer cells cultured in the absence of fibroblasts. Significantly, clonogenic growth of the primary breast cancer cells was also significantly reduced or abolished when the tumor cells were co-cultured with fibroblasts that had been pre-treated with a fixed dose of butein.We conclude that fibroblasts pre-treated with non-toxic doses of butein  no longer support the clonogenic growth of small numbers of primary breast cancer cells seeded into co-cultures. These results suggest that interference with the interaction between fibroblasts and breast cancer cells by the natural herbal compound, butein, should be further investigated as a novel experimental approach for possibly suppressing the growth of micrometastases of breast cancer. Rhus verniciflua Stokes. In Far Eastern countries such as Korea, Japan, and China, the compound has been traditionally used for treatment of pain, thrombotic disease, gastritis, stomach cancer, and parasitic infections [Butein  was passaged in Leibovitz's medium supplemented with 15% fetal calf serum. Normal fibroblasts  obtained from the breast of a 65 year old female were passaged at 37°C in minimal essential medium (Eagle's) supplemented with 2 mM L-glutamine, Earle's balanced salt solution (1.5 grams/Liter), sodium bicarbonate, 0.1 mM non-essential amino acids, 1 mL sodium pyruvate, and 10% fetal calf serum in a 5% COWe seeded 100 UACC-812 breast cancer cells into individual wells of a 96-well cell culture plate containing a confluent monolayer of fibroblasts growing in minimal essential growth medium supplemented as described above. At intervals of 3–4 days, fresh medium was added. After 14 days, the cells were fixed with 70% ethanol for 10 minutes prior to staining for 3 minutes with 0.1% toluidine blue. The wells were then washed with distilled water, and the numbers of colonies of tumor cells containing eight or more confluent cells were counted using inverted microscopy. Each experiment was performed in triplicate, and the means and standard deviations for each treatment and control group were then compared using a two-tailed, unpaired t-test.A monolayer of normal fibroblasts was seeded with 100 UACC-812 tumor cells/well containing complete culture medium (control) or medium supplemented with various modulators of fibroblast function. The fibroblast modulators that we tested included troglitazone . The tissues were carefully minced into small pieces and then digested overnight at 37°C in collagenase II  solution in cell culture medium. Epithelial cells and organoids were then isolated by differential centrifugation of the digest , washed,Co-culture of 100 UACC-812 human breast cancer cells on a monolayer of human fibroblasts without butein treatment typically yielded 50–75 distinct colonies of easily recognizable tumor cells on a background of fibroblasts  does not significantly reduce fibroblast viability [Clonogenic growth of small numbers of breast cancer cells in vivo ,6 as weliability  and is aiability ,2. MoreoRhus verniciflua Stokes are directly cytotoxic in vitro to human colon adenocarcinoma cells and lymphoma cells [Previous studies have shown that higher concentrations of butein extract from ma cells ,14. Butema cells . The mecma cells . Butein ma cells , tyrosinma cells , suppresma cells , inhibitma cells , and inhma cells .Our results suggest that butein may also suppress the growth of tumor cells through a second, indirect mechanism that does not involve direct toxicity to the tumor cells themselves. At this time, however, the precise molecular mechanisms by which butein interferes with the interaction between fibroblasts and breast cancer cells remain undefined.Clonogenic growth of small numbers of breast cancer cells co-cultured with fibroblasts pre-treated with butein is markedly reduced. These results suggest that butein can inhibit the growth of tumor cells through an indirect mechanism that interferes with the interaction between fibroblasts and breast cancer cells. Our results also suggest that the herbal compound butein should be further investigated as a potentially useful experimental approach for suppressing the growth of small numbers of breast cancer cells in early micrometastases.PPAR-peroxisome proliferation activator receptorCOX-cyclooxygenaseThe authors declare that they have no competing interests.MKS conceived and designed the study, performed the experiments with the primary tumor cells, analyzed the data from all experiments, and wrote this report. JT and GC performed the other experiments and reported the data.The pre-publication history for this paper can be accessed here:


A man in a suit and bowler hat walks awkwardly down the street, each convoluted step a labored movement. He lifts up one knee, then briefly stoops. Stepping forward, he swings the other leg out to the side then kicks high in the air. In this old Monty Python skit, the man works for the Ministry of Silly Walks. It's his job to walk this way. The rest of us, however, tend to stroll along—or throw baseballs, or lift coffee mugs—in a much more efficient manner.There's a nearly infinite number of silly walks, throws, and lifts, but somehow people tend to settle on one best way of doing these things. However, scientists studying motor control have been hard pressed to figure out what exactly we're doing when we move. People may be striking a balance between sloth and speed: too slow and our throws lack oomph; too fast, and instead of dunking our donuts in our coffee, we dunk our whole fist. Or people might be minimizing some version of jerk—physicists' and engineers' term for changes in acceleration.  But so far, such models that start by assuming people minimize error or jerk haven't allowed researchers to deduce what dictates how people move.To help solve this recalcitrant problem, Konrad Körding and colleagues, as reported in PLoS Biology, took a page from economists, who have long used equations called utility functions that incorporate the costs and benefits of a situation. Say you like oranges better than apples, but oranges cost more. Given a certain budget for fruit, the utility function says how many of each you should buy. Similarly, Körding and colleagues observed people's preferred movements, then inferred an underlying utility function that presumably describes bias in the nervous system for different movements.To see which movements people preferred, the researchers engaged people in a simple virtual reality system. The subjects moved a joystick that fought back: it was connected to a set of motors that produced varying forces—with a strong force for a short time, say, or a mild force for a longer time. Over and over, the subjects moved their cursors from one spot to another. After each pair of moves, the subjects then chose which of the two movements they found easier.In this way, the researchers were able to rank a large set of different movements relative to each other by individuals' preferences. They found a surprising amount of agreement among the subjects on which movements were preferable. They also got a counterintuitive result: as the duration of the resistance got longer, people actually preferred stronger resistance. The researchers speculate that subjects didn't mind larger resistance when it acted over a longer period because the force takes longer to ramp up to its maximum value. Subjects would have more time to adjust—just as when someone gradually pushes into you, you can stay standing by leaning into them, whereas if they shove you with the same force it can knock you off balance.By showing that utility functions can be of use not only in explaining the marketplace but also motor control, Körding and colleagues have added a new tool to biologists' repertoire. Though their approach hasn't closed the case on the mysteries of movement, it could help explain why we settle for a particular, non-silly walk.


Cluster sample study designs are cost effective, however cluster samples violate the simple random sample assumption of independence of observations. Failure to account for the intra-cluster correlation of observations when sampling through clusters may lead to an under-powered study. Researchers therefore need estimates of intra-cluster correlation for a range of outcomes to calculate sample size. We report intra-cluster correlation coefficients observed within a large-scale cross-sectional study of general practice in Australia, where the general practitioner (GP) was the primary sampling unit and the patient encounter was the unit of inference.Each year the Bettering the Evaluation and Care of Health (BEACH) study recruits a random sample of approximately 1,000 GPs across Australia. Each GP completes details of 100 consecutive patient encounters. Intra-cluster correlation coefficients were estimated for patient demographics, morbidity managed and treatments received. Intra-cluster correlation coefficients were estimated for descriptive outcomes and for associations between outcomes and predictors and were compared across two independent samples of GPs drawn three years apart.Between April 1999 and March 2000, a random sample of 1,047 Australian general practitioners recorded details of 104,700 patient encounters. Intra-cluster correlation coefficients for patient demographics ranged from 0.055 for patient sex to 0.451 for language spoken at home. Intra-cluster correlations for morbidity variables ranged from 0.005 for the management of eye problems to 0.059 for management of psychological problems. Intra-cluster correlation for the association between two variables was smaller than the descriptive intra-cluster correlation of each variable. When compared with the April 2002 to March 2003 sample  the estimated intra-cluster correlation coefficients were found to be consistent across samples.The demonstrated precision and reliability of the estimated intra-cluster correlations indicate that these coefficients will be useful for calculating sample sizes in future general practice surveys that use the GP as the primary sampling unit. Cluster sample study designs are a cost-effective way of sampling difficult to reach populations. Examples include sampling schools to obtain cluster samples of students or medical practitioners to sample patients. ClusterBoth the size of the intra-cluster correlation and the number of observations sampled within each cluster influence the power of the study. Even for a small intra-cluster correlation, as is often found in general practice and community samples, the loss of power can be appreciable, particularly if the size of the cluster is large,4.Estimates of the size of intra-cluster correlations come from post hoc examination of studies that have used either allocation or sampling by cluster and a number of intervention studies have published observed intra-cluster correlation coefficients -6. Many Intra-cluster correlation varies within a study and depends on the outcome under analysis,4,6. TheIn one Australian study Carlin and Hocking examinedThis paper reports some of the intra-cluster correlations observed in the Bettering the Evaluation and Care of Health (BEACH) program, a large cross-sectional survey of general practice patient encounters in Australia, where a random sample of general practitioners was used as the primary sampling unit.The BEACH study draws a new random sample of Australian general practitioners (GPs) each year, and this provided an opportunity to assess the stability of intra-cluster correlation coefficients across successive samples. If a population is re-sampled using the same cluster survey design, will the intra-cluster correlation coefficient for a particular outcome be the same across samples? This analysis takes an applied approach, examining the observed intra-cluster correlations for a range of demographic, morbidity and treatment outcomes.The BEACH program is a continuous study of general practice activity commenced in 1998. The BEACH method is described in detail elsewhere and a brief summary is reported below.A random sample of approximately 1,000 general practitioners (GPs) is drawn each year from the Health Insurance Commission's sampling frame of the population of GPs in Australia. The GP population is randomly ordered into a list and GPs are recruited sequentially from the list, with re-randomisation of the sampling frame every three years,12. GPs A single page encounter form contains elements including:• Patient age and sex.• Whether English was the main language spoken at home.• Whether the patient holds an Australian health care concession card.• The problems managed by the GP at the encounter (up to four problems per encounter).• Treatments received at the encounter, including medications, other procedures, referrals and orders for pathology and imaging tests.Although sample weights are calculated each year for population estimates, the outDescriptive outcomes were defined as rates, means and percentages of single variables, e.g. mean age, per cent of encounters with female patients, per cent of encounters where at least one respiratory problem was managed. BEACH samples the GP-patient encounter, not independent patients. If a patient returns to the GP in the sampling period then that patient contributes two (or more) encounters to the sample. Therefore BEACH estimates are not true "prevalence" rates because the denominator, the population of GP-patient encounters, is many times larger than the population of all general practice patients. To avoid misunderstanding in this paper we have used the term "descriptive" rather than "prevalence" to report single variable estimates and their accompanying intra-cluster correlation coefficients. Descriptive rates are interpreted for example as "Proportion of patients at encounter who are female".Demographic variables include patient sex, patient age, whether the patient held a health care concession card and whether the main language spoken at home was not English.Problems were classified using the International Classification of Primary Care (ICPC-2). The uppTreatment outcomes included the proportion of encounters that resulted in at least one medication, the proportion that received at least one referral, the proportion that received at least one order for an imaging test and the proportion receiving at least one order for a pathology test.Intra-cluster correlation coefficients were calculated for associations between variables using logistic regression e.g.: the effect of patient age (predictor) on the rate of cardiovascular problems (outcome).Obtaining the sample size for cluster designs involves calculating the sample size under the assumption of simple random sampling and then inflating the number of observations to allow for the design effect of the cluster sample. The design effect (Deff) of an outcome has been defined as the ratio of the variance taking into account the cluster sample design and the variance of a simple random sample (srs) design with the same number of observations.(clustersample)/Variance(srs)Deff = VarianceIntra-cluster correlations and their standard errors for the outcome variables were calculated using the method described by Carlin & Hocking. SpecifiThe intra-cluster correlation coefficient (ICC) was then calculated from the design effect using the formula:ICC = (Deff - 1)/(k - 1)and the approximate standard error (SE) of the intra-cluster correlation was calculated using the formula,6:m = number of clusters, k = mean number of observations per cluster.where The intra-cluster correlations and respective 95% confidence intervals for the second BEACH year sample from the period April 1999 to March 2000 were compared against those in the year 5 sample (April 2002 to March 2003) to assess whether the intra-cluster correlations were consistent across samples over time. All calculations specified the GP as the primary sampling unit.From April 1999 to March 2000, 1,047 GPs were recruited, recording a sample of 104,700 patient encounters. From April 2002 to March 2003, 1,008 GPs were recruited and 100,800 encounters recorded. Table The two samples of patient encounters were similar in terms of demographics Table  In the yFor descriptive estimates of demographic variables the intra-cluster correlation ranged from 0.055 for sex of patient at encounter to 0.451 for language spoken by the patient at home. Table . With a For descriptive estimates of the management rates of morbidity problems, the intra-cluster correlations ranged from 0.005 for estimates of eye problems to 0.059 for estimates of psychological problems, with design effects of between 1.5 and 6.8 respectively.The intra-cluster correlation coefficients for treatments received ranged from 0.028 for any imaging tests ordered to 0.056 for any medications.For bivariate relationships between an outcome and predictor, the association ICCs were considerably smaller than the descriptive ICCs Table . This paWhen analysing the association between cardiovascular problems as the outcome and selected demographic variables, the ICCs ranged from 0.042 (patient language as the predictor) to 0.003 (patient sex as the predictor) declare that they have no competing interests.SK conceived the research questions, undertook the analysis and wrote the main draft of the manuscript. PC participated in formulating the research questions and the design of the analysis, undertook a literature search and assisted in the writing of the main draft and subsequent revisions of the manuscript.The pre-publication history for this paper can be accessed here:


The SARS coronavirus is the etiologic agent for the epidemic of the Severe Acute Respiratory Syndrome. The recent emergence of this new pathogen, the careful tracing of its transmission patterns, and the ability to propagate in culture allows the exploration of the mutational dynamics of the SARS-CoV in human populations.We sequenced complete SARS-CoV genomes taken from primary human tissues , cultured isolates , and five consecutive Vero cell passages  arising from SIN2774 isolate. These represented individual patient samples, serial in vitro passages in cell culture, and paired human and cell culture isolates. Employing a refined mutation filtering scheme and constant mutation rate model, the mutation rates were estimated and the possible date of emergence was calculated. Phylogenetic analysis was used to uncover molecular relationships between the isolates.th October 2003, including 22 from patients in Singapore, revealed the mutations engendered during human-to-Vero and Vero-to-human transmission as well as in multiple Vero cell passages in order to refine our analysis of human-to-human transmission. Though co-infection by different quasipecies in individual tissue samples is observed, the in vitro mutation rate of the SARS-CoV in Vero cell passage is negligible. The in vivo mutation rate, however, is consistent with estimates of other RNA viruses at approximately 5.7 × 10-6 nucleotide substitutions per site per day (0.17 mutations per genome per day), or two mutations per human passage (adjusted R-square = 0.4014). Using the immediate Hotel M contact isolates as roots, we observed that the SARS epidemic has generated four major genetic groups that are geographically associated: two Singapore isolates, one Taiwan isolate, and one North China isolate which appears most closely related to the putative SARS-CoV isolated from a palm civet. Non-synonymous mutations are centered in non-essential ORFs especially in structural and antigenic genes such as the S and M proteins, but these mutations did not distinguish the geographical groupings. However, no non-synonymous mutations were found in the 3CLpro and the polymerase genes.Close examination of whole genome sequence of 54 SARS-CoV isolates identified before 14Our results show that the SARS-CoV is well adapted to growth in culture and did not appear to undergo specific selection in human populations. We further assessed that the putative origin of the SARS epidemic was in late October 2002 which is consistent with a recent estimate using cases from China. The greater sequence divergence in the structural and antigenic proteins and consistent deletions in the 3' – most portion of the viral genome suggest that certain selection pressures are interacting with the functional nature of these validated and putative ORFs. The Severe Acute Respiratory Syndrome (SARS) was first reported in November 2002 and rapidly spread to a number of distant global regions by early 2003. A new coronavirus, the SARS-CoV, was identified to be the cause of SARS ,2 and waPreviously, our analysis of 14 SARS sequences (five of which originated from Singapore) in May 2003 indicated that there are two different genotypes circulating in the world . Recentl). The RNA genome templates were converted into double strand cDNA and sequenced as previously described [).SARS-CoV from the primary patient tissues were isolated by homogenizing the tissues in PBS buffer followed by a low speed centrifugation to obtain the viral particle containing supernatant. The virus-containing samples were also inoculated into Vero cell E6. The cells were maintained at 37°C using the usual viral cell culture media, and repassaged after 7 days of incubation. The virus-containing supernatants of homogenize or different passages of Vero cell E6 showing CPE were centrifuged at 23,000 RCF for 2.5 hours to pellet the viral particles and followed by RNA extraction using the QiAmp viral RNA mini kit  were further confirmed using a sensitive Mass Spectrometry based genotyping assay that was developed within our institute . The RNAWe aligned the 54 SARS-CoV genomes using CLUSTALW . To miniq be the rate of noisy mutation in a genome . The probability that, at a given nucleotide, a noisy mutation is shared by exactly i out of m different isolates is . Thus, the probability that a given nucleotide position has an erroneous mutation shared by more than k isolates is . In a genome with n bases, applying the Bonferroni inequalities, the probability that at least one position is corrupted by noise more than k times is p ≤ n × s. In the case of 54 SARS-CoV genomes analyzed in this paper, , m = 54, k = 2, n = 30000, and hence the p-value of mutations that satisfy the filter is ≤ 2.2 × 10-4.The significance of the variations that pass the proposed mutation filter  can be assessed by calculating the probability that a random noise would meet the filtering criterion. The null hypothesis is that the noisy variations are generated independently between genomes. Let d found in an isolate from its ancestor is proportional to the mutation rate k and the temporal difference t between the isolate and its ancestor, so that d = k × t. Based on the Singapore patients contact tracing information  and the time difference (t) for each pair, and estimate the mutation rate k for the model using least square fitting. The goodness-of-fit were measured using the adjusted R-square statistics.In the estimation of SARS-CoV daily mutation rate, we employed the conservative constant mutation rate model , where tdx = d0 + kx, where k is the daily rate of mutation, x is the sampling date measured relative to 1st November 2002, and dx is the number of mutations, as compared to the civet cat isolate, of the isolate sampled at date x. Twelve data points were calculated and used to fit the model. The date of origin can be solved by solving x for dx = 0. The goodness-of-fit was measured using the adjusted R-square statistic.Another pertinent question in the analysis of SARS-CoV evolution is prediction of the possible date of origin of the human SARS-CoV. Based on the animal-origin hypothesis of SARS-CoV, we assumed the SARS-CoV isolated from palm civet cat as the putative principal isolate that infected the human population. Adhering to the constant mutation rate model, we fit the following model: in vitro cell culture are negligible.First, we sought to determine the rate of mutation of the SARS-CoV in Vero cell culture. To achieve this, we subjected SIN2774 isolate to 5 passages in Vero cells. At the appearance of cytolysis at each passage samples were withdrawn and their genomes completely sequenced. Any ambiguities by capillary sequencing were clarified by mass spectroscopic validation . Our resNext, we asked whether the transition from human tissue to growth in Vero cell culture engendered either mutations or clonal selection. The SARS-CoV were sequenced from three human tissue-Vero cell culture pairs of viral samples from Singapore and one pair was obtained from the public domain . The results shown in Table Singapore encountered an unusual incident where a stable lab SARS-CoV isolate commonly used for in vitro experimentation accidentally infected a laboratory worker . We sequ-5, or 2.25 errors per SARS-CoV genome. Accordingly, we suspected that sequencing errors in the reported SARS sequences would be approximately 1–2 bases per reported genome. We used this information to assess the true sequence variants reported in the public SARS sequence databases employing a "mutation filter" [p ≤ 2.2e - 4) as compared to the probability of finding a false mutation shared by more than one isolate (p ≤ 0.19). These results are consistent with our error estimates as outlined above.Inferring phylogenetic relationships between the known SARS-CoV isolates using existing public data has been problematic because of the potential for sequencing errors. Moreover, the rate of SARS-CoV mutation in culture was not previously known and was thought to be significant given the mutation rates in other RNA viruses. Our experiments provided information as to the potential causes and rates of sequence variations of the SARS-CoV in culture. Based on our Vero cells passages and human-to-Vero transition data, we estimated that, at most, one sequence variation from the original tissue virus can be accounted for by in vitro culture artifacts. The average base-calling error probability (as reported by PHREP ) of our We tested the biological validity of this approach by examining the mutational frequency of known genes in the SARS genome. Because of the importance of the 3CL protease and the polymerase for viral replication, we suspected that true non-synonymous mutations in the SARS-CoV present in clinical samples might be rare in these two ORFs in comparison to other structural genes such as those encoding the S, M, and N proteins. Without a mutation filter, sequence variations are commonly observed in the 3CL protease and the polymerase genes. However, when mutations are identified only as variants seen in two or more isolates, then no mutations are detected in the critical 3CL protease and polymerase genes, whereas mutations are noted in the S, M, and N genes regardless of the filter stringency Figure . TherefoUsing this filter stringency, we assessed the phylogenetic relationship between all 54 isolates describing the recent SARS epidemic. TOR2, Urbani, SIN2500, HKU-39849, CUHK-Su10 formed the core of the early isolates. Employing these as "root", four major clusters appeared: two Singaporean branches, one Taiwan branch, and one North China branch . The rates for synonymous and non-synonymous mutations were equivalent at 2.5 × 10-6 and 3.2 × 10-6 nucleotide substitutions per site per day respectively. Using the Singapore isolates with known date of onset, and using the SZ3 and SZ16 genomes isolated from palm civet cat [-6 nucleotide substitutions per site per day, (adjusted R-square = 0.5880) and the estimated possible "date" of SZ3/SZ16 emergence was Oct 21, 2002. Overall, the mutation rate of SARS-CoV appears to be consistent with the reported rate of other viruses [We obtained the precise dates of symptom onset of 13 Singaporean cases (Table S1). Using the common mutations identified through application of the mutation filter, we employed the constant mutation rate model and estimated the mutation rate of the SARS-CoV during this recent epidemic. We estimated the mutation rate to be 0.1722 nucleotides per day, or 5.7 × 10ivet cat  as the pThe mutational analysis also revealed 5 separate deletions and one insertion that distinguished the different isolates , cases reported in North China, and cases found in Taiwan. Moreover, we show that these molecular sequence associations can be effectively used to more precisely reconstruct contact tracing. Our estimated the daily substitution rate of SARS-CoV to be 0.1722 nucleotides, or 5.7 × 10 viruses ,14. Taki[et. al. , examininsortium , examininsortium  projecteNone declared.VBV and ETL performed most of the data analysis and prepared the draft manuscript. STSY and LAE provided SARS-CoV samples from patients and Vero cells. KFT and OEE provided continue SARS-CoV containing Vero cell passage samples. YR and LWS designed the experiments and coordinated sample acquiring and the viral genome sequencing. CLW and TZ generated all DNA sequence data. WHL processed all DNA sequence and assembled the viral genomes. JL verified all sequence variations by conducting MALDI-TOF mass spectrometry analysis. PK assessed the effects of nucleotide variations to ORFs and their proteins. VBV and PML carried out the mathematical and statistical analysis.The pre-publication history for this paper can be accessed here:Table S1 List of the sequences used in the analysis. This list is available at http://giscompute.gis.a-star.edu.sg/sars_mut_dyn/Click here for fileTable S2 Single nucleotide variations (SNVs) detected initially in capillary sequencing (second row) and subsequently confirmed by MALDI-TOF MS-based genotyping (first row). This table is available at http://giscompute.gis.a-star.edu.sg/sars_mut_dyn/Click here for fileTable S3 Complete list of single nucleotide variations (SNVs) observed in the 54 SARS-CoV isolates. The Singapore sequences used were all based on capillary sequencing. This data is available at http://giscompute.gis.a-star.edu.sg/sars_mut_dyn/Click here for file


Recently, a global commitment has been made to expand access to antiretrovirals (ARVs) in the developing world. However, in many resource-constrained countries the number of individuals infected with HIV in need of treatment will far exceed the supply of ARVs, and only a limited number of health-care facilities (HCFs) will be available for ARV distribution. Deciding how to allocate the limited supply of ARVs among HCFs will be extremely difficult. Resource allocation decisions can be made on the basis of many epidemiological, ethical, or preferential treatment priority criteria.Here we use operations research techniques, and we show how to determine the optimal strategy for allocating ARVs among HCFs in order to satisfy the equitable criterion that each individual infected with HIV has an equal chance of receiving ARVs. We present a novel spatial mathematical model that includes heterogeneity in treatment accessibility. We show how to use our theoretical framework, in conjunction with an equity objective function, to determine an optimal equitable allocation strategy (OEAS) for ARVs in resource-constrained regions. Our equity objective function enables us to apply the egalitarian principle of equity with respect to access to health care. We use data from the detailed ARV rollout plan designed by the government of South Africa to determine an OEAS for the province of KwaZulu–Natal. We determine the OEAS for KwaZulu–Natal, and we then compare this OEAS with two other ARV allocation strategies: (i) allocating ARVs only to Durban  and (ii) allocating ARVs equally to all available HCFs. In addition, we compare the OEAS to the current allocation plan of the South African government . We show that our OEAS significantly improves equity in treatment accessibility in comparison with these three ARV allocation strategies. We also quantify how the size of the catchment region surrounding each HCF, and the number of HCFs utilized for ARV distribution, alters the OEAS and the probability of achieving equity in treatment accessibility. We calculate that in order to achieve the greatest degree of treatment equity for individuals with HIV in KwaZulu–Natal, the ARVs should be allocated to 54 HCFs and each HCF should serve a catchment region of 40 to 60 km.Our OEAS would substantially improve equality in treatment accessibility in comparison with other allocation strategies. Furthermore, our OEAS is extremely different from the currently planned strategy. We suggest that our novel methodology be used to design optimal ARV allocation strategies for resource-constrained countries. A mathematical model that allows to determine a strategy for distribution of limited medical resources  such that each individual in need will have an equal chance of receiving treatment The HIV/AIDS epidemic is having a devastating impact in sub-Saharan Africa and other resource-constrained regions. Recently, the World Health Organization and other organizations have committed to expand access to antiretrovirals (ARVs) in the developing world, the United States government has pledged to provide $15 billion for AIDS in Africa and the Carribean, and drug prices have fallen . It has Here, we use operations research to address this important resource allocation problem and to design ARV allocation strategies that are rational and equitable. The allocation decisions that we make here are based on ethical criteria, and not on epidemiological or preferential treatment priority criteria. Specifically, we determine the optimal allocation strategy that would ensure that each individual with HIV has an equal chance of receiving ARVs. We present a novel spatial mathematical model of treatment accessibility that we use in conjunction with an equity objective function to determine an optimal equitable allocation strategy (OEAS) for ARVs in a resource-constrained region. We quantify how changing the size of the catchment region surrounding each HCF, and the number of HCFs utilized for ARV distribution, alters the OEAS. Specifically, we use data from the detailed ARV rollout plan designed by the government of South Africa to determine an OEAS (based upon a variety of assumptions) for the province of KwaZulu–Natal. We also discuss how our proposed ARV allocation strategy differs from the currently proposed plan.Our current analysis is applied to the South African province of KwaZulu–Natal, although our methodology could be applied to any resource-constrained setting. KwaZulu–Natal is the largest province in South Africa with a population of approximately 9.4 million and has more people infected with HIV than any other province  as a weighting function that determines the treatment accessibility to a HCF based upon distance i,j,d and iI as the number of people with HIV in community i. The distance, ij,d between community i and HCF j is based on the longitude (lon) and latitude (lat) of each location and is determined byWe assume that the number of people with HIV who will travel to a specific HCF is directly proportional to the number of individuals with HIV in that particular community, but that the probability of an individual traveling to receive ARVs  decreases with distance from the HCF. We define R is the radius of the earth, taken to be 6,371 km, and the angles are in radian measure. We calculate the “effective demand” of community i on HCF j to be the number of people with HIV in community i that will travel to HCF j for ARV regimes, namely, fiI. Thus, demand on HCFs for ARVs is reduced by the treatment accessibility function. Our model is conceptually similar to the “gravity” models that have been used to predict retail travel [f(d) = exp(−kd2), where k is a dispersal length scale parameter determining the radius of the catchment region. The size of the actual catchment regions is unknown, but based upon distances from communities to HCFs in KwaZulu–Natal (see k = 0.003786). We vary the catchment region by considering a 20-km radius (k = 0.0151) and a 60-km radius (k = 0.00168). The different catchment regions that we simulate  for each HCF are illustrated in where l travel , plan lal travel , and detl travel . Howeveratal see A we assu= exp−kd, where kj,j, D to beTo determine how many ARVs should be allocated to each HCF, we first calculate how a given supply of ARVs will be distributed from each HCF to the surrounding communities in the catchment region. We calculate the “effective demand” on HCF j (where there are m communities). Then, we model the distribution of ARVs from a HCF to each community within the catchment region as the proportion of the “effective demand” on HCF j that is contributed by the respective community. Accordingly, ARVs will be distributed from HCF, j, to each community as the ratiowhich sums the “effective demand” of all communities on HCF i by the drug supply allocated to HCF j isTherefore, the number of people treated in community jS is the number of regimes allocated to HCF j. Hence, the total number of people with HIV treated in community i,i,T summing over all n HCFs iswhere A ARV regimes for a total of We establish an equity objective function to determine the optimal equitable allocation of ARVs to each HCF so that all individuals with HIV have an equal chance of receiving treatment. To obtain the same fraction of treated individuals in each community, given that there are E, by solving for the number of ARVs to be allocated to each HCF , whilst enforcing the following three constraints: (i) ensure that the total number of ARVs available is equal to the sum of the supply allocated to all HCFs,Our goal is to minimize jS ≥ 0, j = 1…n); and (iii) ensure that the number of people treated in each community is not greater than the number of people with HIV in the community . We note that if a different objective is required, then all of our preceding analysis still holds and only the functional form of the objective function needs to be altered. To solve the problem, and determine the OEAS, we used successive linear programming operations research techniques [(ii) ensure that only a positive number of ARVs are allocated to each HCF (chniques .k = 0.003786). The proportion of infected individuals that are treated at each location is displayed graphically in E = 0.27 for our OEAS, compared with (i) E = 0.50 and (ii) E = 133.88 for the comparison allocation strategies. There is large diversity in the fraction of individuals with HIV treated per community when equal quantities of ARVs are given to each HCF, evidenced by an inter-quartile range of 0.025%–41.746% compared with inter-quartile ranges of 0%–0% and 0.011%–9.982% for the first comparison strategy and our OEAS, respectively. Therefore, equal access is not obtained if equal quantities of ARVs are allocated to each HCF. Obviously, allocating to only one HCF (the first comparison strategy) could also be considered unequal because although the inter-quartile range is minimal, effectively only one community (Durban) receives ARVs. Our OEAS, while not perfect, achieves the best equality possible given the accessibility constraints and limited ARV supply.The OEAS of ARVs in KwaZulu–Natal that we determined is complex see A and 3B.The catchment region for HCFs is a factor of large uncertainty. We considered three catchment region sizes: radii of 20 km, 40 km, and 60 km. We also simulated two additional cases with increased numbers and locations of HCFs  should be used. The strategy that we are advising may be fairly easy to accomplish at the policy level because the health-care infrastructure  already exists, although consideration must be made for issues such as the training and transportation that is necessary, which may be costly. In contrast, increasing the size of catchment regions may be very difficult. Obviously, increasing both the number of HCFs and the size of the catchment region each services would substantially increase equality of access to health care in KwaZulu–Natal.Future modeling studies could extend our work by not making the simplifying assumption that all patients have similar ease of travel over the same distance and by including weighting functions on distance impedance for different communities  . Here, we have shown how to calculate optimal ARV allocation strategies based upon the principle of equity. Future research is necessary to compare ARV allocation strategies based upon the principle of efficiency  in order to determine whether utilizing different principles for optimization would result in similar (or different) allocation strategies.The World Health Organization and the Joint United Nations Programme on HIV/AIDS have identified three core principles that should underlie the effort to fairly distribute ARVs, namely: urgency, equity, and sustainability . They stAlthough we have focused on one equitable strategy, there are many other ARV allocation strategies that are ethical. Uneven access to HIV treatment has the very real potential to fracture social and political structures and could lead to intrastate and/or interstate conflict . GovernmTreatment priority decisions for individuals could be based on many different criteria, including disease progression , socioeconomic status, ethnicity, and who is thought to have the greatest risk of transmitting infections . Although it could be argued that behavioral core groups should be targeted to receive ARVs because this may have the greatest epidemiological impact, such an allocation strategy would be neither feasible nor practical to implement. For example, sex workers are an obvious behavioral core group, but many women would likely claim to be sex workers if they knew that ARVs were only available to sex workers. Additionally, the ethics of targeting such groups in favor of other societal groups must be questioned. It could also be argued that, to maximize the preventative effect of ARV therapy, ARVs should be concentrated in virological core groups  ,26; thisOur model has been applied to the South African province of KwaZulu–Natal, but it can be applied by government health officials in any resource-constrained country. In many of the countries worst affected by the HIV pandemic, scarcity of resources will mean that not everyone that could potentially benefit from ARVs will be able to access them. Many of the decisions that must be made to develop an effective response to the HIV/AIDS epidemic are inevitably underpinned by ethical considerations. Leadership in most resource-constrained regions cannot avoid these decisions. Whilst there has been considerable attention given to South Africa, many other countries worldwide either have plans in place  or are in the process of developing national programs for ARV distribution through the public health system  . LegitimAntiretroviral drugs can change the lives of patients with HIV/AIDS. Their high price, however, means that many poor countries do not have enough of these drugs to treat all the people who need them. The decision of who will get treatment is very difficult, and different ways to come up with ethical solutions to the problem have been proposed.One of the approaches is to try to make sure that every infected person has the same chance to get antiretroviral drugs. David Wilson and Sally Blower, the authors of this study, wanted to find a scientific strategy to achieve this goal of equal access.They used mathematical models to calculate how to distribute available drugs among hospitals and doctor's offices so that each patient in a particular area had an equal chance to get treated.When they used their approach on a real example, the South African province of KwaZulu–Natal, they found that making some changes to the current plans for drug distribution would lead to more equal access among all of the individuals with HIV in the province. Instead of only 17 out of the 54 health care facilities in KwaZulu–Natal distributing the drugs (which is the current plan of the South African government), Wilson and Blower calculate that it would be fairer if all 54 facilities distributed the medicines.Mathematical models like the one used here are always based on assumptions and simplifications. As a consequence, they are never perfect matches for a real-life situation, but they can help to guide complicated decisions. This article suggests that the approach Wilson and Blower developed could help to determine strategies for equitable allocation of limited HIV treatment resources.The authors hope that the tools they developed will be used by policy makers in resource-poor countries to guide their strategies. They are keen to work with these policy makers to adapt and optimize the method to local settings and priorities.http://www.who.int/hiv/pub/advocacy/en/ethicsmeetingreport_e.pdfReport by the World Health Organization and the Joint United Nations Programme on HIV/AIDS on ethics and equitable access to HIV/AIDS treatment: http://www.who.int/ethics/en/background-macklin.pdfRuth Macklin's report on ethics and equity in access to HIV treatment: http://www.who.int/ethics/en/background-pro-poor3.pdfThe Pro-Poor Health Policy Team's report on priority in HIV/AIDS treatment: http://www.who.int/bulletin/volumes/82/1/en/news.pdfNews article from the World Health Organization Bulletin on the South African HIV/AIDS treatment program:


Molecular markers for localized colon tumours and for prognosis following therapy are needed. Proteomics research is currently producing numerous biomarker studies with clinical potential. We investigate the protein composition of plasma and of tumour extracts with the aim of identifying biomarkers for colon cancer.By Surface Enhanced Laser Desorption/Ionisation – Time Of Flight / Mass spectrometry (SELDI-TOF/MS) we compare the protein profiles of colon cancer serum with serum from healthy individuals and the protein profiles of colon tumours with normal colon tissue. By size exclusion chromatography, we investigate the binding of HNP 1-3 to high mass plasma proteins. By microflow we investigate the effect of HNP 1-3 on mammalian cells.Human Neutrophil Peptides -1, -2 and -3 (HNP 1-3), also known as alfa-defensin-1, -2 and -3, are present in elevated concentrations in serum from colon cancer patients and in protein extracts from colon tumours. A fraction of HNP 1-3 in serum is bound to unidentified high mass plasma proteins. HNP 1-3 purified from colon tumours are lethal to mammalian cells.HNP 1-3 may serve as blood markers for colon cancer in combination with other diagnostic tools. We propose that HNP 1-3 are carried into the bloodstream by attaching to high mass plasma proteins in the tumour microenvironment. We discuss the effect of HNP 1-3 on tumour progression. The diagnostic stage of colon cancer determines survival. Patients diagnosed with localized tumours have a 75% probability of 5 year survival, whereas patients diagnosed with distant metastases only have a 5–10 % probability of 5 year survival . The samples were centrifuged at 14,000 rpm for 10 minutes and the pellet was discarded (repeated twice). The tissue protein extracts were stored at -80°C until use. Pilot studies were performed on different chips (data not shown) and the NP20  (Ciphergen) chip was chosen for the tissue screening. NP20 chips were placed in Bioprocessor (Ciphergen) and pre-treated with 50 ul tissue binding buffer  for 5 minutes on shaker (250 rpm) (repeated twice). 5 ul tissue protein extract was diluted in 50 ul tissue binding buffer and incubated in Bioprocessor on NP20 chips for 40 minutes at room temperature on shaker (250 rpm). Spots were washed twice in 250 ul tissue washing buffer  for 5 minutes. The chips were air dried for 10 minutes, followed by treatment with two times 0.6 ul 100% sinapinic (SPA) matrix solution. SPA was obtained from Ciphergen in 5 mg aliquots and dissolved ) immediately before the screenings.Colon cancer serum samples were obtained from patients before surgical treatment. Normal serum was obtained from a group of healthy individuals matched by age and gender to the cancer patients. All serum samples were stored at -80°C until use. Serum pilot studies were performed on different chips to monitor the presence of HNP 1-3 in serum (data not shown). The immobilised metal affinity capture (IMAC30) chip was chosen for the actual screening and was pre-treated with nickel before analysis: 5 ul 100 mM NiSO4 were added to each spot and left on shaker (250 rpm) for 5 minutes (repeated twice). The chips were placed in Bioprocessor and incubated with 100 ul MQ for 5 minutes on shaker (250 rpm). Each spot was treated with 50 ul serum binding buffer  and left on shaker for 5 minutes (250 rpm). Serum samples were thawed on ice and 1 ul serum was diluted in 50 ul serum binding buffer and applied to spots and left on shaker (250 rpm) at room temperature for 40 minutes. The sample solution was removed and the spots were washed twice in 200 ul serum washing buffer , followed by one wash in 200 ul MQ water. The chips were removed from the Bioprocessor and left to air dry for 20 minutes followed by treatment with two times 0.6 ul SPA.Only freshly made matrix solutions were used and the instrument was calibrated daily. Cancer and normal samples were run side by side. The chips were analysed on a PBS II instrument (Ciphergen). All spectra in each screening were normalised based on total ion current.4HCO3, pH 8,0) was added, and the chip was left for 10 hours at 40°C in humidity chamber after which the chip was left to air dry for 20 minutes. 1 ul CHCA matrix (prepared as the SPA matrix solution) was added and the peptide map was analysed on the PBS II instrument. Peptide maps of trypsin autodiggest were used as controls. Identification was done with the PepIdent software on the Expasy server. For the reduction experiment, HNP 1-3 were first denaturation by heating (10 minutes at 80°C) followed by treatment with DTT  and the peptides were incubated on an NP20 chip and analysed on the PBS II instrument.100 ul protein extract from colon tumour tissue in tissue lysis buffer was loaded unto a RP-HPLC column  in buffer A (0.065% TFA in MQ-water) and proteins were eluted in a gradient of 0–100% buffer B (0.05% TFA in acetonitrile (ACN)). Elution of peptides was monitored by absorbance at 280 nm. All protein-containing fractions were analysed by Matrix Assisted Laser Desorption/Ionisation-Time of flight (MALDI-TOF) on the PBS II instrument: 1.5 ul fraction was incubated with 0.6 ul SPA on a Gold array (Ciphergen) and left to crystallise, followed by an additional treatment with 0.6 ul SPA and analysed in the PBSII instrument. The HNP 1-3 containing fraction (32% buffer B) was further purified on a peptide gelfiltration column . Elution of peptides was monitored by absorbance at 280 nm and protein-containing fractions were again analysed by MALDI-TOF. Purified HNP 1-3 were identified by on-chip trypsin digestion: 10 ul of HNP 1-3 fraction was applied to an NP20 chip and left on shaker (250 rpm) at room temperature for 40 minutes. The solution was removed and the spot was washed twice with 10 ul water. In order to denature peptides prior to digestion, the chip was left on heating block (80°C) for 5 minutes. The chip was cooled on ice for 2 minutes. 10 ul trypsin digestion solution . Elution of peptides was followed by absorbance at 280 nm. All protein containing fractions were analysed by MALDI-TOF on PBS II (Ciphergen) as described above. Maximum signal intensity of 40 individual peaks was plotted as a function of elution volume and an approximate elution curve was calculated.For micro flow experiments, canine kidney cells (MDCK cells) were plated onto poly-d-lysine coated cover slips at a concentration 3000 cells/well, grown in Dilbeccoo's Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) for five days with the result of confluent islands. Microflow was performed in an Eppendorf micromanipulator 5171 and transjector 5246 system mounted on a Leica DMIRBE inverted research microscope. Micro capillaries  were pulled to an outer diameter of 0.85 nm on a Sutter P-97 Micropipette Puller. The dye-loaded cells were visualized by excitation at 470 nm and recorded at 509-nm emission using Haupage version 3.3.18038 software and Kappa CF 15/4 MC-S camera (Leica). The MDCK cells were recorded (in CO2 independent media) on the inverted DMIRBE inverted research microscope. The capillary was placed 20 nmover the confluent cells with a constant flow (1300 hPa). The MDCK cells were exposed to peptide and calcein (20 mM) fractions for 60 minutes.-16).We performed pilot studies of colon tumour and normal colon tissue on a variety of chips with different chemical properties and with different binding and washing conditions. Based on these preliminary studies, we found that the expression of three peptides with mass/charge ratio (m/z) values of 3372, 3443 and 3486 , were upregulated in the tumour samples. The three peptides were visible on different chips and under different binding conditions (data not shown). The strongest signal of HNP 1-3 in tissue extract was obtained on the NP20  chip, whereas the strongest signal of HNP 1-3 in serum was observed on the IMAC30 (Ni) , and these conditions were chosen for the actual screenings. We emphasize that in general the protein profiles of serum and tissue were very different when using the same protein chip for serum and tissue extract. However, individual peaks were present on several types of chips, and observed in both serum and in tissue extract, for example the characteristic triplet with m/z 3372, 3443 and 3486. Protein extract from 40 colon tumour and 40 normal colon tissue samples were analysed on NP20 chips and 125 colon cancer serum samples and 100 normal serum samples were analysed on IMAC30 (Ni) chips. All spectres in each screening were pooled and normalised based on overall ion current. Each spectrum produced approximately 40 to 90 protein peaks in the range from 2 to 80 kDa FIG. . StatistCompared to other peptides in the same range, HNP 1-3 showed average signal intensity in most normal colon tissue extract, whereas the HNP 1-3 signal was extremely high in most tumour samples . In the normal serum samples the HNP 1-3 signals were weak and only slightly stronger in the cancer serum.The markers were purified by RP-HPLC, peptide gelfiltration and on-chip purification, after which they were identified by peptide mapping as HNP-2 (3372 Da), HNP-1 (3442 Da) and HNP-3 (3486 Da) Table . The mas50 ul colon tumour extract in Lysis buffer was applied to a peptide gelfiltration column. Elution of peptides was followed by absorbance at 280 nm. All fractions were analysed by MALDI-TOF on the PBS II instrument (Ciphergen). Maximum signal intensity of 40 individual peaks was plotted as a function of the elution volume and an approximate elution curve was calculated FIG. . HNP 1-3The cytotoxicity of HNP 1-3 purified from colon tumours was tested by exposing MDCK cells to different fractions purified from colon tumours. Calcein were added to the fractions and the solutions were left to flow over the cells for one hour. By fluorescence microscopy calcein was observed to accumulate in cells exposed to HNP 1-3/calcein fractions, whereas cells treated with fractions containing other (unidentified) tissue peptides did not uptake calcein FIG. . FurtherAbnormal concentration of HNP 1-3 in blood has previously been demonstrated in connection with benign conditions. Elevated concentrations of HNP 1-3 following infection  has been found in plasma, blood and other body fluids , and plaHNP expression has previously been linked to different types of tumours and cell lines. HNP-1 has been detected in lung tumours  and in tPrevious studies indicate that HNP 1-3 expression in tumours primarily originate from tumour invading eosinophils  and neutin vitro and that high concentrations of HNPs causes precipitation of plasma proteins; specifically 2-macroglubulin and C1 complement [We explain the elevated concentrations of HNP 1-3 in colon cancer serum by unspecific binding between HNP 1-3 and high mass plasma proteins. We suggest that the peptides attach to plasma proteins in the tumour area and are carried into the bloodstream. The HNP 1-3 we observe in high mass fractions from size exclusion, could also be explained by multimerisation: In one study it was demonstrated that defensins form voltage dependent channels in lipid bilayer membranes and further conductance investigations suggested that the channels were formed by multimers containing 2–4 molecules  and a crmplement ,23 has bmplement  showed tmplement . TogetheCommon to beta-defensin 2 (another member of the defensin family) and HNP 1-3 is an uneven distribution of surface charges. Beta-defensin 2 has been shown to bind to a chemokine receptor . It has in vivo function of HNP 1-3. One study showed that HNP 1-3 mediates lysis of tumours in a concentration dependent manner [-4 M) are cytotoxic for human monocytes, whereas lower concentration of HNP-1 (10-8 to 10-9 M) increases TNF-alpha production by monocytes [in vivo HNP 1-3 purified from colon tumours are capable of lysing MDCK cells. Our study was based on a 60 minutes microflow study. This screening set-up did not allow us to investigate the minimum concentration of HNP 1-3 necessary for lysis.The exact concentration of HNPs in the tumour microenvironment may decide the t manner . This isonocytes . In a stonocytes  it was sThe high concentration of HNP 1-3 observed in tumours and the observation that HNP 1-3 are capable of lysing mammalian cells may lead to the conclusion that the peptides serve to the benefit of the host by killing tumour cells. However, in one study HNP 1-3 were found to bind to HLA-Class II molecules and were capable of reducing the proliferation of a HLA-DR-restricted T-cell line after stimulation  and coulOur results add to the evidence that HNP 1-3 bind to high mass plasma proteins. We suggest that the peptides are released in the inflammatory site and passively diffuse into the blood stream. HNP 1-3 have been observed in primary tumours of different tissues and the peptides are known to play a fundamental role in the innate immune system. We suggest that the peptides may serve as markers for colon cancer in combination with established diagnostic tools and as prognostic markers following therapy.This study was partly financed by Colotech ltd. and partly by Glostrup Hospital. Jakob Albrethsen, Rikke Bøgebo and Hans Raskov receive salary from Colotech A/S.Steen Gammeltoft, Jesper Olsen and Benny Winther receive salary from Glostrup Hospital.JA performed the SELDI-TOF/MS screenings, the protein identification experiments and the size exclusion study. RB did the statistical analysis, BW did the microflow study, JO obtained the biological samples. HR and SG planned the project.The pre-publication history for this paper can be accessed here:


Bacteria are particularly adept at making themselves indispensable by insinuating themselves into some fundamental aspect of an organism's biology. The endosymbiotic hypothesis proposes that this is how certain eukaryotic organelles evolved from endosymbiotic bacteria. Insights into the mechanisms governing endosymbiosis will help biologists understand how this mutually beneficial relationship evolved and provide clues to one of the fundamental questions in biology: How did the eukaryotic cell evolve?When it comes to exploiting a niche, endosymbionts take the prize. In endosymbiosis, one organism—the Buchnera aphidicola, and discover clues to the molecular foundation of their shared fate. Over 10% of insect species rely on endosymbionts for their development and survival. In this issue, David Stern and colleagues look at one of the most studied pairs, the pea aphid and Buchnera are transferred from clusters of bacteriocytes in the mother to the adjacent early-stage embryo, it has been unclear how the bacteriocytes develop. Previous studies of the bacteria's genome have failed to explain the genetic basis of Buchnera's ability to invade aphid cells. Consequently, Stern and colleagues have focused on the bacteriocytes, the specialized insect cells that house Buchnera, shedding light on the development of these cells as well as on the evolutionary adaptations in the aphid that made the bacteriocytes hospitable to Buchnera.While it is known that Buchnera's presence in the cell, and they identify three aphid transcription factors (proteins that regulate gene expression) that are expressed in three distinct stages during early-bacteriocyte development in the aphid embryo. The first protein is expressed just before Buchnera enters the embryo; a second, as the bacteria invades; and a third, after the transfer is nearly complete. A second wave of the same transcription factors occurs at a later stage in aphid embryo development and increases the population of bacteriocytes.The researchers show that bacteriocytes differentiate and proliferate independently of Buchnera-carrying aphid species, appears to be an evolutionarily conserved feature of aphids. It even occurs in an aphid species that once had a Buchnera endosymbiont and now has a yeast-like symbiont that lives outside the bacteriocytes. But this process is not observed in males of another aphid species that do not carry Buchnera. While traces of the first transcription factor activated in bacteriocytes are evident, the characteristic gene-expression pattern is not, and the aphids have no mature bacteriocytes.This two-step specification of bacteriocytes, which occurs in related Buchnera involved significant adaptations by the host.While it seems that the aphid has evolved new domains of expression in the bacteriocyte for these transcription factors—none of these transcription factors is expressed at a similar stage in other insects—the researchers cannot yet say whether these genes direct the specification of bacteriocytes. Still, these transcription factors are likely to play important roles in the bacteriocyte, suggesting that the union of aphids and


We investigated patient perspectives on switching from injection to oral B12 therapy.Although oral replacement with high doses of vitamin B12 therapy. One hundred and thirty-three patients who receive regular B12 injections were included from three family practice units  in Toronto.This study involved a quantitative arm using questionnaires and a qualitative arm using semi-structured interviews, both to assess patient views on injection and oral therapy. Patients were also offered a six-month trial of oral B12. In a multivariate analysis, patient factors associated with a "willingness to switch" to oral B12 included being able to get to the clinic in less than 30 minutes , and believing that frequent visits to the health care provider  or the increased costs to the health care system  were disadvantages of injection B12. Fifty-five patients attempted oral therapy and 52 patients returned the final questionnaire. Of those who tried oral therapy, 76% (39/51) were satisfied and 71% (39/55) wished to permanently switch. Factors associated with permanently switching to oral therapy included believing that the frequent visits to the health care provider  and travel/parking costs  were disadvantages of injection B12. Interview participants consistently cited convenience as an advantage of oral therapy.Seventy-three percent (63/86) of respondents were willing to try oral B12 is both feasible and acceptable to patients. Oral B12 supplementation is well received largely due to increased convenience. Clinicians should offer oral B12 therapy to their patients who are currently receiving injections, and newly diagnosed B12-deficient patients who can tolerate and are compliant with oral medications should be offered oral supplementation.Switching patients from injection to oral B However, because approximately 1% of orally ingested B12 is absorbed via simple diffusion throughout the gastrointestinal tract [12 deficiency [Intramuscular injections of vitamin Bficiency -12.12 therapy would decrease physician burden, increase patient control over therapy, and avoid patient discomfort and inconvenience. Switching patients from B12 injections to oral therapy would also result in savings to the health care system [12 therapy [12.Oral Be system . While s therapy -15, litt12 are willing to switch to oral therapy and to explore the reasons for their choice.Efforts to switch patients who are well established on parenteral therapy (and who have previously been told they require lifelong injections) may fail without an understanding of what factors influence patient acceptability of oral therapy. Therefore, we combined qualitative and quantitative methods to test the hypothesis that patients offered oral B12 deficiency, offered the option of a six-month trial of oral replacement, and administered a follow-up questionnaire. Throughout the study, semi-structured interviews were conducted for the qualitative arm. We received ethics approval from the Sunnybrook and Women's College Health Sciences Centre Research Ethics Board. All patients provided informed consent.We administered a questionnaire to patients with B12 deficiency. We excluded patients if their last injection occurred more than three months before the start of patient recruitment, if they had left the practice, if their B12 therapy had been discontinued or if they had already switched to oral therapy, if they did not speak English and did not have access to a translator, or if they had been recently diagnosed with a serious illness. After applying these criteria, 133 patients were included in the study , regardless of age and etiology of B12 therapy to injections: "Studies have shown that vitamin B12 pills are just as effective and safe as B12 injections." Educational sessions on the effectiveness of oral therapy were held with the nursing staff (since they administer the injections) at all sites, and they were encouraged to pass this information on to their patients. As well, contact information for the study investigators was provided to the patients in case they had any questions.The recruitment cover letter included one sentence describing the equivalency of oral B12 injection. The initial questionnaire elicited demographic data, medication history, logistical aspects of B12 related visits, history of past B12 therapy, and attitudes about injection and oral therapy. Non-responders received up to two telephone reminders.We placed the initial questionnaire see   in pati12 tablets (one 1000 μg tablet daily), and were offered testing of serum B12 levels at baseline (one month after their last injection) and after the six-month trial. Patients had the option to withdraw from the trial and return to injections at any time for any reason. A follow-up questionnaire was placed in the charts of patients who tried the pills. This questionnaire re-assessed patients' attitudes about the different forms of B12 therapy and asked whether they would continue on oral supplementation or return to injection therapy  as the cut-off for inclusion of individual patient factors into multivariate logistic regression models. We then performed backwards stepwise regression to determine statistically significant relationships after adjustment. We generated both crude and adjusted odds ratios with 95% confidence intervals.Where variables were not already dichotomous , we dichotomized the variable of interest . To ascertain the relationship between questionnaire responses and preference for oral therapy (as "willingness to switch" and "permanently switching") we performed bivariate and multivariate analyses. We used a p-value of 0.20 . Amongst the 17 participants, four were selected from those not willing to switch, while of the 13 who were willing to try oral therapy, six were interviewed prior to, two during, and five after the trial of oral therapy.One investigator (D.C.) conducted 17 semi-structured interviews in a private meeting room at the family practice unit. A purposive sample was selected amongst those willing to be interviewed to reflect diversity in terms of sex, age, willingness to switch, and final choice of therapy after the trial of B12 therapy, his or her relationship with health care providers, perceived advantages and disadvantages of both pills and injections, and attitudes about past and current B12 therapy. Analyses of the transcripts were performed independently by two investigators (J.K. and D.T-K.), using a three-step content analysis approach to identify and collate relevant themes [The interviews were audiotaped and transcribed. They lasted 20–30 minutes, with questions about the patient's knowledge and history of their Bt themes . ThroughFor our initial questionnaire, we received responses from 86 out of the sample of 133 patients, for a response rate of 64.7%. Selected characteristics of the study population are presented in Table 12 therapy, number of non-B12 related visits per month, improvement in perceived well-being since initiating B12 injections, several perceived disadvantages of injections , and several perceived disadvantages of pills .Sixty-three of our 86 respondents reported a willingness to switch to oral therapy. A large number of patient factors had no clear statistical relationship to a "willingness to switch" to oral therapy  and were excluded from the multivariate model: age, education, income, drug insurance coverage, study site, number of prescription medications, mode of travel to the clinic, years of past B12 injections, several perceived disadvantages of injections , and one perceived disadvantage of pills (won't see MD/nurse as often). Factors associated with willingness to switch after multivariate adjustment were: being able to get to the clinic more quickly  , and believing that injection therapy is disadvantageous due to the need for frequent visits to health care provider  and the increased costs to the health care system   chose pills.Fifty-two patients returned the follow-up questionnaire. Over three-quarters (39 of 51 respondents) reported being satisfied or very satisfied with oral therapy. Only 8% of patients (4/51) perceived that they felt worse with pills, while 23.5% (12/51) felt better and the rest felt the same. Self-reported compliance was good; 48 patients or 92% reported forgetting to take the pills two times or less per month. Thirty-nine patients  stated that they wished to permanently switch to B12. Patient factors that on bivariate analyses had a p-value of less than 0.20 and were consequently included in the multivariate model were: patient beliefs that frequent visits to see the doctor or nurse and the associated travel/parking costs are disadvantages of injections, and the belief oral B12 would add unnecessarily to an already large number of prescribed oral medications. Table Again, many patient factors had no association with a desire to permanently switch to oral B12 levels were obtained for 39 of the 55 patients who switched to pills . Only one patient's serum level decreased but even she remained within the normal range.Baseline and post-intervention serum BThe mean age of interview participants was 71 (range 54–92) years, with 7 men and 10 women.12 injections they had been receiving in the past were effective: "I find it gives me more energy." Patients who expressed this view were often not willing to switch to oral therapy, or if they did try the pills, they eventually went back to injections. Others felt that the injections hadn't helped much: "There is a little feeling that, 'Oh, I'm taking this. I should feel better,' but I'm not sure if I do." These patients tended to switch permanently to oral therapy.Some patients felt that the BWhen asked about the anticipated efficacy of oral therapy, some patients, particularly those who were not willing to switch, were sceptical: "Well, I don't think it will work. If the doctors suggested that I needed to go on the injections, I'm certainly pretty sure that the pills are not going to work. Otherwise he'd have put me on pills, would he not?" Many others were unsure about the efficacy of oral therapy.12 pill. Other advantages of oral therapy included savings to the health care system and ease of travel. Cited disadvantages of injections included decreased compliance due to the need for frequent visits and potential complications associated with injections. A number of patients decided to switch because they were interested in participating in a research study. Some patients acknowledged that oral therapy would benefit those who are averse to needles, however, needles did not bother most of the patients interviewed: "...needles don't bother me, because – well, I don't know. They just don't seem to bother me. I know some people are very concerned about it."Nearly every patient cited convenience as an advantage of switching to B12 and for three of the five patients interviewed afterward, all of whom permanently switched to oral therapy, there was a high level of satisfaction with oral B12. One patient even reported feeling better on oral B12: "Well, I'm more level. I don't feel at the end of the month that I'm running out of energy. I'm quite well aware of that."  and the other cited decreased energy . . see .12 is both feasible and acceptable to patients. Of those who responded to our initial questionnaire, nearly three-quarters were willing to try oral B12, and of those who did switch, most were satisfied and the majority wished to remain permanently on oral therapy. These patients believed that injections are disadvantageous because they are associated with too many visits to their health care provider and with higher patient costs (in terms of travel and parking expenses). In the qualitative arm of our study, most patients cited convenience as the reason for wanting to switch from injections to pills. We were also able to confirm findings from other studies indicating that oral B12 is biochemically equivalent to, if not better than, parenteral therapy.We found that switching patients from injection to oral BOne non-intuitive finding from the initial questionnaire assessing the factors associated with trying oral therapy Table  was that12 levels  and again 6 months after oral therapy, we observed that one patient was a non-responder , and 4 patients had serum levels that increased but remained below 295 pmol/L (a level considered by some to be borderline B12 deficient)[12 levels and, if available, functional tests such as serum homocysteine and methlymalonic acid. The sub-optimal response in these patients may be due to patient non-compliance or sub-optimal dosing  [By monitoring trough serum Beficient). This re et al.) . FurtherThe primary strength of our study was that we achieved triangulation through the use of both quantitative and qualitative methods. By using questionnaires, we were able to provide estimates such as switch rates and levels of patient satisfaction; with the qualitative arm, we were able to more thoroughly explore patient perspectives.12. However, the sample was large enough to identify a number of patient factors associated with switching from injection to oral therapy and to determine that patient attitudes toward oral B12 therapy are greatly influenced by its convenience. A second limitation was that we did not assess the knowledge and attitudes of our patients' physicians and nurses, as they may have influenced patient's expectations and perceptions regarding the effective of oral B12 therapy. Finally, another limitation was the potential participant bias, evidenced by the differences between responders and non-responders in terms of age and sex. However, among the responders, neither age nor sex was significantly associated with switching to oral therapy, suggesting that any differences between participants and non-participants were likely irrelevant.The main limitation of our study was the relatively small sample size for the quantitative arm, which led to large confidence intervals for the estimates of the relationship between patient factors and switching to oral B12 deficiency and providing educational sessions to encourage discontinuing injections, 38% stated they would leave the practice if denied their injections [While there have been several studies examining physicians' perspectives on switching from intramuscular to oral therapy -21, we kjections . Explana12 therapy may be acceptable for most patients, it may not be appropriate for those who will not be compliant with oral medications, such as patients with significant memory impairment or cognitive dysfunction, unless they have caregivers who could ensure compliance. Oral therapy also may not be appropriate for those with swallowing difficulties; for such patients, sublingual B12 therapy, which has been shown to be equally effective, may more appropriate [While oral Bropriate .12 therapy to their patients who are currently receiving injections if they can tolerate and are compliant with oral medications. Most patients who switch from parenteral to oral therapy are satisfied and wish to stay permanently on oral therapy. They often cite the inconvenience and the travel-related expenses associated with frequent visits to the doctor as disadvantages of parenteral therapy. Therefore, oral B12 therapy is generally well-received by patients, and should be considered by clinicians as a superior alternative to the traditional injections for most patients.In summary, the results of our study suggest that clinicians should offer oral BThe author(s) declare that they have no competing interests.JK designed and coordinated the study, analyzed the qualitative and quantitative data, and drafted the manuscript. DC conducted the in-depth interviews and assisted in data collection. ID helped to develop the questionnaire and performed statistical analyses of the quantitative data. DTK analyzed the qualitative data. RU conceived the study and provided guidance on all aspects of the project. All authors participated in the development of the manuscript and gave approval to its final submission for publication.The pre-publication history for this paper can be accessed here:Initial questionnaireClick here for fileFollow-up questionnaireClick here for fileReasons for switching to oral therapyClick here for fileReasons for not switching to oral therapyClick here for file