Adherence to oral antidiabetic medications is often suboptimal. Adherence differences may contribute to health disparities for black diabetes patients, including higher microvascular event rates, greater complication-related disability, and earlier mortality.In this longitudinal retrospective cohort study, we used 10 years of patient-level claims and electronic medical record data (1/1/1992–12/31/2001) to assess differences in short- and long-term adherence to oral antidiabetic medication among 1906 newly diagnosed adults with diabetes  in a managed care setting in which all members have prescription drug coverage. Four main outcome measures included: (1) time from diabetes diagnosis until first prescription of oral antidiabetic medication; (2) primary adherence (time from first prescription to prescription fill); (3) time until discontinuation of oral antidiabetic medication from first prescription; and (4) long-term adherence (amount dispensed versus amount prescribed) over a 24-month follow-up from first oral antidiabetic medication prescription.Black patients were as likely as whites to initiate oral therapy and fill their first prescription, but experienced higher rates of medication discontinuation  and were less adherent over time. These black-white differences increased over the first six months of therapy but stabilized thereafter for patients who initiated on sulfonylureas. Significant black-white differences in adherence levels were constant throughout follow-up for patients initiated on metformin therapy.Racial differences in adherence to oral antidiabetic drug therapy persist even with equal access to medication. Early and continued emphasis on adherence from initiation of therapy may reduce persistent racial differences in medication use and clinical outcomes. Diabetes is a leading cause of death and disability in the United States, costing an estimated $174 billion in lost productivity and health care expenditures each year. ,3 LandmaRacial differences in medication adherence may reflect disparities in other components of diabetes care such as patient education and counseling, insurance coverage, and geographic access to health care services. -16 In prThe purpose of this study was to determine whether racial differences in short-term and long-term adherence exist in this setting where a strong focus is placed on preventive services, coordination of care, and standardization of services and where other possible determinants of suboptimal adherences to medication have been reduced, including economic barriers in access to care. Given our previous findings that racial differences in glycemic control and self-monitoring of blood glucose exist and persist in spite of equal access to services and treatment quality, we expected to find similar race-related differences in medication adherence, with blacks hypothesized to be consistently less adherent to prescribed regimens than whites over time.Harvard Vanguard Medical Associates (HVMA) is a large multi-specialty care group comprised of 14 health centers serving over 300,000 people of diverse ethnic and socioeconomic backgrounds in the Boston area. Using HVMA's electronic medical record system, we captured patient-level data from 1992–2001 from all ambulatory and inpatient encounters, including pharmacy encounters and laboratory test results, diagnoses, procedures, and therapies. The electronic medical record system also contained data on a patient's date of birth, gender, months of membership, and census tract of residence (which was linked to socioeconomic characteristics of the neighborhood such as income and education using the 1990 census files). In addition, all records for emergency room visits, hospitalizations, and other services received outside the HVMA system were recorded in a linked claims database. Prescriptions for oral antidiabetic medications were extracted from HVMA electronic ambulatory encounters and linked to corresponding pharmacy medication dispensing claims data. Previous studies have documented the reliability of these longitudinal data. ,23To be included in the study, patients had to be at least 18 years old, of black or white race, and newly diagnosed with diabetes. Race, the key independent variable, was determined from clinician reports. Although information on race was not uniformly collected, a study comparing self-reported and medical record data on race classification found a 96% agreement (unpublished data). Patients were classified as newly diagnosed based on a record of ≥ 1 inpatient diagnosis of diabetes [ICD-9 codes 250.XX] or ≥ 2 outpatient diabetes diagnoses, or ≥ 1 prescription or dispensing of insulin or oral antidiabetic medication , with no previous record of a diabetes diagnosis or medication. Patients had to be continuously enrolled (with no more than 45 days of disenrollment) in HVMA and insured with Harvard Pilgrim Health Care (HPHC) for at least one year prior to and one year after the first prescription of oral antidiabetic medication.Due to the difficulty in measuring insulin adherence because of the wide variation of insulin dosages across patients, adherence measures for patients on insulin treatment were not developed. Patients who were prescribed or dispensed an insulin therapy at any point in the follow-up period were excluded from the study cohort. We also excluded patients with a diagnosis of polycystic ovarian syndrome  unless they also received a diabetes diagnosis. Any patient with a diagnosis of gestational diabetes during the observation period was also excluded. Since less than 1% of all study patients initiated therapy on meglatinide, thiazolidinedione, or alpha-glucosidase inhibitors, we excluded them in the main analyses and focused on the vast majority of patients whose initial therapy was sulfonylurea or metformin. The resulting study sample consisted of 1906 patients.We used four outcome measures to capture patients' patterns of medication use. First, we examined the time from diabetes diagnosis until the first prescription for oral antidiabetic medication. Second, among those with prescriptions, we examined primary adherence by measuring the time from first prescription for an oral antidiabetic drug until the patient filled the prescription. Third, we measured the time between first prescription and discontinuation of medication therapy (= 60 days without a refill following last day without medication). Lastly, we examined long-term adherence, defined as the monthly average rate of adherence, comparing dose available in each month through prior dispensings in relation to dose prescribed in each month over a 24-month follow-up from time of first prescription. To construct these adherence measures, we assessed refill-based medication adherence linking prescribing information from clinical encounter data and pharmacy claims for dispensed data. Standard refill-based medication adherence measures assume that days supply is equivalent to daily dose and, therefore, cannot distinguish between physician initiated changes in therapy and patient noncompliance.  Our measOur covariates were comprised of both baseline (defined as 12 months prior to the beginning of a patient's medication treatment) and time-varying covariates. Baseline covariates included gender, age, census block group-derived median neighborhood household income and average educational attainment level, body mass index (BMI), and average HbA1c level over the prior year. For time-varying covariates, we used a previously validated method  to calcuWe performed all statistical analyses using SAS V8.02 .  Chi-squaGiven the clinical differences in side-effects between sulfonylurea and metformin and the greater likelihood in discontinuation of metformin therapy due to its side-effects, we stratified all analyses by these two most commonly prescribed oral therapy groups. Patients on a combination of metformin and other oral antidiabetic medication were represented in the metformin group.We conducted survival analyses to examine racial differences in the time to initiation and discontinuation of oral antidiabetic drug therapy. Within the oral antidiabetic drug therapy initiators, we produced unadjusted cumulative probability curves of time to first prescription since diagnosis, time to filling this first prescription, and time to discontinuation of medication use, all stratified by race. We fit Cox survival regression models, ,28 with For our final multivariate analyses, we estimated the effect of race on long-term adherence among patients for 24 months, allowing patients who discontinued use for more than 60 days but reinitiated therapy thereafter to contribute to these analyses. We excluded patients with fewer than 24 months of follow-up from the time of first prescription or who switched or augmented medication therapy with insulin during the 24-month follow-up. The resulting analytical sample for the adherence analyses included 1404 patients. Within therapy groups, we used log-linear models  to estimate and test the difference in mean adherence rates for the two race groups at eight quarterly time intervals. In these models, we controlled for key demographic and clinical covariates as well as clinical sites serving disproportionately high numbers of black patients. Quarterly intervals were included in the model as dummy terms (with the initiation month of drug treatment as a referent). To test whether race differences varied across quarters, we used likelihood ratio tests. We also stratified these analyses by pre-treatment HbA1c group   to adjusAs shown in Table Figure We found no black-white differences in primary adherence to medication for either drug therapy group. Over 90% of black and white patients filled their first prescription within 30 days. By the end of the 24-month follow-up, nearly all patients had filled their prescription at least once.As shown in Figure Figure After controlling for key covariates, blacks were consistently less adherent to sulfonylurea medication than whites, with significant black-white differences occurring within the second quarter of follow-up  among newly diagnosed diabetes patients. However, rates of discontinuation in medication use varied by race, with blacks discontinuing sooner and at higher rates than whites, particularly among patients initiating sulfonylurea therapy. Re-initiation of therapy after discontinuing for more than 60 days did not appear to show a substantial increase in adherence levels for either race group over time. In concordance with our hypothesis, blacks were consistently less adherent to oral antidiabetic drug therapy than whites over time.Our findings agree with previous studies reporting that blacks have lower adherence to oral antidiabetic medication. However,While our findings demonstrate persistent racial differences in adherence levels for patients on sulfonylurea or metformin therapies, racial differences in patterns of medication use varied by drug therapy group. For those taking sulfonylureas, the largest reductions in adherence and the separation in adherence levels between black and whites occurred early in treatment (quarter 2–3 following initiation of therapy); racial differences appeared to decline somewhat thereafter. This modest narrowing of race differences over time may result from patients reinitiating their medication therapy after early discontinuation. Among metformin patients, race differences in adherence, while somewhat inconsistent, appear to increase over time. Additional research is needed to understand longitudinal variations in adherence, timing of initiation of insulin or re-initiation of oral antidiabetic drug use following discontinuation, and racial differences in these behaviors.In a recent study,  we foundThis study has additional potential limitations worthy of discussion. First, because race was determined from clinician reports, misclassification may have occurred. However, assessments of white and black race from these sources have been shown to be highly reliable. ,23 Race/We controlled for a number of other potentially race-related barriers to adherence, such as the number of physician visits per month. However, by doing so, we may have over-controlled for racial disparities, such that we underestimate the effect of race on adherence. For example, if differences in adherence are due to blacks' use of fewer services or receipt of less patient education, controlling for those factors may reduce estimates of the impact of race on adherence behavior. For future work, a focus on the volume and nature of patient interactions with the health care system may enable us to identify additional determinants of racial disparities and avenues for intervention.The rates of missing data for HbA1c and BMI in Table The age of our data represents a decade when sulfonylureas were prescribed over metformin as the first line of treatment contrary to ADA recommendation. However, over time, metformin has become more commonly prescribed as the initial oral therapy over sulfonylureas, particularly among obese patients. While the rate of initiating sulfonylureas and metformin reported in this study may not represent the current prescribing behavior of providers, self-management guidelines have not changed since our study data and the implications of these study findings remain very relevant to current medical practice.Finally, our findings are restricted to a single multi-specialty care group in Massachusetts. While we cannot generalize our findings to non-managed care settings, the large proportion of black patients enrolled at HVMA allows for comparison to other studies of racial/ethnic differences in diabetes self-management in similar settings.Racial differences in adherence to oral antidiabetic drug therapy persist over time within a health system that provides uniform access to services and high quality of care for diabetes patients. While differences do not appear in initiation of medication, racial gaps are quickly evident in discontinuation and adherence over time. Early and continued emphasis on sustaining medication use after initiation of medication may lead to improved adherence to antidiabetic medication. Aggressive, culturally-specific promotion of adherence among blacks or implementation of a patient-centered approach to identify barriers to adherence and provide self-care supports during early treatment may potentially reduce persistent racial differences in self-management practice and clinical outcomes.The authors declare no competing interests including specific financial interests and relationships and affiliations relevant to the subject of this manuscript.Substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data . Substantial involvement in drafting the manuscript or revising it critically for important intellectual content . Provision of final approval of publishable version The pre-publication history for this paper can be accessed here:


Agents targeting the egfr-mediated signalling pathway are increasingly part of the therapeutic armamentarium for the treatment of advanced lung, head-and-neck, and colorectal carcinoma. The egfr inhibitors (egfris) approved in Canada include the tyrosine kinase inhibitors erlotinib and gefitinib (in selected cases), and the monoclonal antibodies (mAbs) panitumumab and cetuximab. Although egfris have been proven effective in the treatment of a variety of malignancies, the entire class of agents is associated with a high prevalence of dermatologic side effects, most commonly skin rash. This reversible condition requires intervention in approximately one third of patients. A proactive, multidisciplinary approach to management can help to improve skin rash and optimize clinical outcomes by preventing egfri dose reduction or discontinuation. In addition, effective management and patient education may help to alleviate the significant social and emotional anxiety related to this manageable side effect, thus resulting in improved quality of life. The present article focuses on egfr-targeted mAbs for the treatment of gi malignancy, addressing the pathophysiology, clinical presentation, and incidence of skin rash caused by this class of agents. Recommendations aimed at establishing a framework for consistent, proactive management of skin rash in the Canadian setting are presented.The epidermal growth factor receptor ( The receptor consists of an extracellular ligand-binding domain, a transmembrane region, and an intracellular tyrosine kinase domain The epidermal growth factor receptor (gi) malignancies, egfr is overexpressed egfr may result in uncontrolled cell growth, proliferation, and angiogenesis, and is associated with a poorer prognosis, manifested by increased metastatic potential and poorer overall survival (os) times egfr is an ideal target for antitumour therapy. Inhibitors of this target can be broadly classified as either tyrosine kinase inhibitors (tkis) or monoclonal antibodies (mAbs), both of which can produce significant skin toxicity.In many solid tumours, including most gastrointestinal : cetuximab and panitumumab  is a recombinant human/mouse (chimeric) immunoglobulin G1 mAb that is administered once weekly. Cetuximab in combination with irinotecan  is indicated in Canada for the treatment of egfr-expressing mcrc and with non-mutated [wild-type (wt)] KRAS after disease progression on fluoropyrimidine-, oxaliplatin-, and irinotecan-containing chemotherapy regimens Panitumumab  is a fully-human immunoglobulin G2 mAb administered once every 2 weeks. Panitumumab is indicated as monotherapy for patients with egfr-targeted mAbs have demonstrated efficacy in the treatment of advanced colorectal malignancy in a number of clinical trials  control group . No significant difference was observed in os, likely because of the high percentage (76%) of bsc patients who crossed over to panitumumab at disease progression. Objective response was observed in 10% of patients randomized to panitumumab and in 11% of patients crossing over to this agent, as compared with 0% in the bsc group In a randomized phase iii study examined the influence of KRAS mutation status on the therapeutic efficacy of panitumumab. That analysis reported that progression-free survival (pfs) was significantly greater in patients with wt KRAS than in patients with a mutant gene (p < 0.0001). Median pfs in wt KRAS patients was 12.3 weeks for the panitumumab group as compared with 7.3 weeks for the bsc group. To account for potential tumour-ascertainment bias in favour of the bsc arm, an interval-censored sensitivity analysis was performed in which radiologic event times were moved to the closest assessment time pre-specified in the study protocol. That analysis showed median pfs times of 16 weeks and 8 weeks with panitumumab and bsc respectively . No significant difference in os was observed between treatment arms for all patients  or between KRAS groups  pfs, the efficacy of panitumumab monotherapy for mcrc is limited to patients with wt KRAS tumours. Accordingly, KRAS mutation status must be evaluated to optimize selection of patients with mcrc for panitumumab monotherapy.A retrospective analysis of the phase iii National Cancer Institute of Canada (ncic) trial that randomized chemotherapy-refractory mcrc patients to cetuximab monotherapy or bsc. Compared with bsc alone, cetuximab treatment was associated with significant improvements in pfs  and os . Median os in the cetuximab group was 6.1 months as compared with 4.6 months in the bsc group ncic trial did not allow crossover to active therapy for patients initially randomized to receive bsc alone.The efficacy of cetuximab monotherapy was evaluated in a phase KRAS mutation status on os and pfs. The authors reported that the effect of cetuximab was significantly greater in the wt KRAS group than in the mutant gene group both for pfs (p < 0.0001) and for os (p < 0.01). No significant difference in os as a function of KRAS status (wt vs. mutant) was observed in the bsc arm . The authors concluded that KRAS mutation status is a strong predictive biomarker and that mutation analysis can be considered a new standard of care in the selection of patients for egfr-targeted therapy KRAS status was available for 69% of patients in the phase iii cetuximab monotherapy study as compared with 92% of the patients in the phase iii panitumumab monotherapy analysis discussed earlier A Cox model analysis of the study examined the predictive effect of ii trial of third-line therapy for patients with mcrc: combination therapy was compared with cetuximab alone. Median time to progression was significantly greater in the combination arm (4.1 months) than in the monotherapy arm (1.5 months). Tumour response rates (22.9% vs. 10.8%) and median survival times were also significantly higher in the combination arm than in the monotherapy arm Cetuximab plus irinotecan was studied in a randomized phase egfr-targeted mAbs excludes many of the severe side effects commonly observed with cytotoxic chemotherapy. As a class, however, egfr inhibitors (egfris) are characterized by cutaneous adverse effects, most commonly a papulopustular reaction involving skin egfr therapy The toxicity profile of egfris at some point during therapy because of skin rash, and up to 32% of physicians reported discontinuing egfri treatment altogether egfri-mediated skin rash may help to maximize benefit for patients by minimizing the negative effects on quality of life and maintaining an optimal mAb dose.In a post-approval survey, 76% of respondents reported holding The next section of this article reviews the pathophysiology, clinical presentation, and incidence of skin rash resulting from mAb therapy in colorectal cancer and presents a practical treatment algorithm summarizing recommendations—from a Canadian perspective—for the management of this skin toxicity. Also addressed are the importance of proactive management, the correlation between rash and efficacy of therapy, and the direction of future research in this area.egfr-targeted mAb is incompletely understood, inhibition of egfr is believed to cause follicular occlusion and rupture because of premature epithelial differentiation and an increase in the expression of genes that stimulate inflammation, apoptosis, and cell attachment Epidermal growth factor receptor is normally expressed in the epidermis, sebaceous glands, and hair follicular epithelium egfri is associated with a spectrum of epidermal-derived toxicities. Although papulopustular skin rash is the most common skin toxicity associated with egfr-targeted mAbs (and the focus of this article), other less frequent side effects can include dry skin, pruritus, fissures, palmar–plantar rash, hyperkeratosis, telangiectasia, hyperpigmentation, blisters, mucositis, and pyogenic granuloma. Changes may also occur to the hair  and nails  Treatment with egfr-targeted mAbs tends to be more severe and to occur with higher incidence than is observed with tkis. It also presents as a more purulent and pustular reaction which may require more aggressive interventions crc treated with egfr-targeted mAbs as third-line therapy, with most cases being mild-to-moderate in severity  version 2.0 Common Terminology Criteria for Adverse Events (nci-ctcae) version 3.0 egfris is questionable nci-ctcae relies heavily on body surface area as a determinant of rash severity. This approach fails to account for the location of egfri-associated rash—generally confined to the face and upper trunk—and also does not consider subjective patient tolerability and discomfort.Accurate grading of egfri-specific grading system, such as that proposed by Pérez–Soler egfri-mediated skin rash, thus allowing clinicians to individually optimize treatment. As well, such a grading system may provide a framework by which various therapeutic interventions may be compared, allowing optimal management strategies to be identified.An egfri-mediated skin rash is based principally on qualitative evidence and anecdotal experience that have yet to be validated by well-controlled randomized clinical trials. Yet, despite the absence of level 1 evidence, a number of consensus statements and algorithms egfri treatment benefits by minimizing the need for significant dose reductions  is the first prospective trial designed specifically to compare primary pre-emption with reactive treatment for egfri-mediated skin toxicity folfiri –based chemotherapy plus every-second-week panitumumab (n = 32) or irinotecan-based chemotherapy plus panitumumab every 3 weeks (n = 26) were randomized to primary pre-emptive or reactive skin treatment. Patients randomized to the pre-emptive treatment arm received daily skin treatment from 24 hours before their first dose of panitumumab through week 6. Patients in the reactive treatment arm received treatment after development of skin toxicity. Skin toxicity treatment in the pre-emptive arm included skin moisturizer, sun-screen, 1% hydrocortisone cream, and doxycycline 100 mg twice daily. Treatment and timing of therapy initiation in the reactive arm were left to the discretion of the investigator. Recently presented results indicated that, as compared with reactive treatment, pre-emptive treatment reduced the incidence of grade 2 or greater skin toxicities by more than 50% without additional side effects. In addition, time to severe skin toxicity was significantly delayed in the pre-emptive treatment arm. The time to first occurrence of any grade 2 or greater skin toxicity was also significantly delayed in the preemptive arm.A controlled study called crc preparing to initiate cetuximab therapy. Patients were also randomized to receive topical tazarotene application to the left or right side of the face. At weeks 1–4 of mAb treatment, the minocycline group (n = 24) had a significantly lower total facial lesion count and a significantly reduced incidence of moderate-to-severe itch as compared with the placebo group . Study authors also concluded that topical tazarotene was not recommended for the management of cetuximab-mediated rash because it provided no clinical benefit and was associated with significant skin irritation egfri therapy were randomized to tetracycline (500 mg twice daily) or to placebo for 4 weeks (n = 61). Although tetracycline did not prevent egfri-induced rash, a reduction in rash severity was observed. At week 4, grade 2 rash was reported in 17% of the tetracycline group and in 55% of the placebo group (p = 0.04). Treatment also improved certain skindex-16 quality-of-life measures, including skin burning or stinging and skin irritation Two randomized double-blind trials have examined the effects of prophylactic skin rash treatment. An 8-week trial studied prophylactic oral minocycline as compared with placebo for patients with megfri-mediated skin toxicities, and to establish a framework for consistent evidence-based treatment approaches based on biologic mechanisms. In addition, further evaluation of patient, nursing, and physician education tools is important and may aid in the promotion of prophylactic intervention, early recognition, and best practices in skin toxicity management, thereby maximizing potential clinical benefit from the egfri class of agents, reducing the risk of serious infection, and improving patient quality of life.Additional well-controlled prospective clinical trials are needed to further examine the potential benefits of primary pre-emption of egfri therapy and with os in mcrc. Data suggest that skin rash may serve as a surrogate marker of egfr-targeted mAb efficacy. In several analyses, both the presence and intensity of skin toxicity predicted objective tumour response in patients treated with cetuximab or panitumumab in mcrc. In patients receiving cetuximab monotherapy for refractory mcrc, longer survival times were observed in patients with rash of any grade as compared with patients experiencing no rash (p = 0.02) ii trial of cetuximab in combination with irinotecan as compared with cetuximab alone in patients with mcrc, response rates in patients with skin reaction were higher than rates in patients without skin reaction  Available retrospective evidence suggests that the appearance and severity of skin rash is positively correlated with objective tumour response to ii and phase iii studies of panitumumab. An exploratory analysis of the pivotal phase iii trial of panitumumab for third-line therapy of mcrc observed a positive correlation between increasing rash severity and improved os  and os  as compared with patients experiencing grades 0–1 skin toxicity iii open-label study in which improved pfs was observed for patients with grades 2–4 as compared with grade 1 skin toxicity Similar results have been observed in phase roved os  9. In a crc receiving egfris who develop skin rash should be treated for the rash while continuing egfr-targeted mAbs, because these patients may derive the greatest clinical benefit from therapy. Note that these observations have yet to be validated by prospective trials, and it should not be assumed that egfr-targeted mAbs are ineffective in patients who do not develop rash.Overall, these observations support the consensus that patients with megfri therapy and have led to the initiation of “dose-to-rash” trials (increasing the dosage until rash is observed) that are examining dose escalation protocols. The phase i/ii dose escalation trial in patients with mcrc with no or slight skin reactions on standard-dose cetuximab treatment, also known as the everest trial, supports the rash-to-survival correlation, with prospective data demonstrating an increase in rash severity and response rate with escalating dose of cetuximabcrc with no or slight skin reactions after 22 days of standard cetuximab therapy were randomized to continue receiving a standard dose or to begin a dose escalation protocol that involved increasing the dose every 2 weeks until a grade 2 skin toxicity or a cetuximab dose of 500 mg/m2 was achieved. As compared with the standard-dose arm, the dose-escalation arm demonstrated an improvement in response rate (risk ratio: 30% vs. 13%). This finding supports the relationship between tumour response rate, egfri dose, and skin toxicity, thereby substantiating the correlation between egfri activity at the level of the egfr receptor on the skin and in the tumour.Retrospectively observed rash-to-survival correlations suggest that individualized dose titration based on the appearance and severity of skin rash may allow for optimization of egfri agent. Accordingly, researchers have speculated that skin toxicity may aid in predicting which patients may preferentially or maximally benefit from egfri therapies. In addition, targeting doses to achieve a desired level of cutaneous toxicity may further increase the efficacy of egfris. Ongoing and planned dose-to-rash studies will further evaluate the potential benefit of dose escalation for patients with mcrc and no or mild skin toxicity from egfr-targeted mAbs. If these studies confirm a relationship between egfri dose and response rate, definitive studies using pfs as an endpoint will be justified Although the mechanism underlying the correlation between skin toxicity and tumour response is currently unclear, investigators have hypothesized that the rash may be a surrogate marker for degree of receptor saturation by the egfr mAbs have demonstrated survival advantages in second- and third-line therapy of mcrc, the use of these agents continues to increase. Forthcoming randomized clinical trial results will also determine whether these agents are efficacious in the first-line setting. As the use of these agents expands in Canada, a better understanding of the associated skin toxicities and optimal patient management strategies is becoming increasingly important and remains a significant area of new educational need for many clinical oncologists, oncology nurses, and general practitioners.Given that anti-The treatment strategies that follow are recommended, based on consensus, for pre-emption and management of mAb-mediated skin rash and other dermatologic toxicities associated with these agents.Patients should be advised to take appropriate sun protective measures, because sun-exposure can exacerbate rash severity on unprotected areas of the body Patients should also be counselled to avoid activities and skin care products that dry the skin. Examples include long, hot showers; alcohol-based or perfumed products; and over-the-counter acne medication. Greasy ointments should also be avoided in favour of frequent moisturizing with alcohol-free emollient creams.For symptomatic relief, oatmeal baths can be soothing and are likely anti-inflammatory Creams are more effective than lotions, and when kept cool , they can provide symptomatic benefit.nci-ctc). Although this grading system is primarily intended for skin rash on the face and scalp, the same grading can be applied to truncal rash, dry skin, pruritus, and nail or periungual changes.Specific Canadian treatment recommendations are presented below according to grade of skin toxicity . Grades egfri therapy Topical 2% clindamycin is recommended for mild-to-severe skin rash, because this antibiotic has demonstrated favourable results, including a drying effect, on inflammatory pustules mediated by The use of a low- to medium-potency topical steroid such as 1% hydrocortisone in a lotion base further enhances treatment of mild-to-severe rash by inhibiting inflammation egfri-mediated skin toxicity does not seem to involve infectious agents, the anti-inflammatory properties of the tetracyclines, via matrix metalloproteinase inhibition, may explain their effectiveness For moderate-to-severe skin toxicity, the oral semi-synthetic tetracycline antibiotics minocycline or doxycycline are recommended in addition to the topical treatments already described. Although egfr therapy.Overall, management of skin rash should be individualized for each patient, depending on the type, severity, and location of the skin toxicity caused by anti-Importantly, several situations warrant patient referral to a dermatologist. Clinicians may wish to refer if the skin toxicity does not improve within 1–2 weeks of treatment. Referral is also recommended if the patient is severely symptomatic  or if multiple hair, nail, and skin issues emerge egfr therapy, including therapy with the egfr-targeted mAbs cetuximab and panitumumab. A proactive, multidisciplinary approach to pre-emption and therapy of skin toxicities will help to limit the incidence of severe symptoms, improve patient tolerance of therapy, maximize quality of life, and optimize clinical benefits of egfr-targeted mAbs by minimizing the need for dose reduction or early treatment discontinuation. The Canadian treatment recommendations and algorithm presented in this article represent the current consensus-derived best practice for treatment of skin rash mediated by egfr-targeted mAbs in patients with mcrc, and they are likely generalizable to other diseases being treated with these agents. Although well-designed prospective trials remain necessary to confirm best practices, educational tools such as this algorithm may serve to guide health care professionals and to inform patients with the goal of maximizing the benefits of egfri therapy. Establishing evidence-based approaches to the treatment of skin toxicities will become an even greater priority as the indications for these agents expand into earlier treatment and adjuvant settings for mcrc and other tumour types.Skin rash is a predictable but manageable side effect of anti-


The clinical utility of a biomarker depends on its ability to identify high-risk individuals to optimally manage the patient. A new biomarker would be of clinical value if it is accurate and reliable, provides good sensitivity and specificity, and is available for widespread application. Data are accumulating on the potential clinical utility of integrating imaging technologies and circulating biomarkers for the identification of vulnerable (high-risk) cardiovascular patients. A multi-biomarker strategy consisting of markers of inflammation, hemostasis and thrombosis, proteolysis and oxidative stress, combined with new imaging modalities  can increase our ability to identify such thombosis-prone patients. In an ideal scenario, cardiovascular biomarkers and imaging combined will provide a better diagnostic tool to identify high-risk individuals and also more efficient methods for effective therapies to reduce such cardiovascular risk. However, additional studies are required in order to show that this approach can contribute to improved diagnostic and therapeutic of atherosclerotic disease. Atherosclerotic disease and its thrombotic complications (atherothrombosis) remain the leading cause of mortality and morbidity in Western society. The mortality associated with atherosclerotic disease is related to the acute coronary syndromes (ACS), including acute myocardial infarction, unstable angina pectoris and sudden cardiac death. There is substantial clinical, experimental and postmortem evidence demonstrating the role of acute thrombosis on disrupted atherosclerotic plaques in the onset of ACS. Atherosclerotic plaque composition, rather than stenotic severity, appears to be central in determining risk of both plaque rupture and subsequent thrombogenicity. Inflammation plays a central role throughout the entire disease progression, and it lies at the root of atherosclerosis and its complications –3. PlaquThere is growing evidence that different types of vulnerable plaques exist that have different functional and biological features. Plaques may have similar structural features and morphologic assessment, but may differ in their biology, their activity, and thus their likelihood of advancing toward clinical complications.In the past, invasive coronary angiography has been the only diagnostic procedure for identifying coronary atherosclerosis. Angiography provides purely morphologic information about vessel lumen diameter. However, various clinical observations in recent years have emphasized the need for more detailed analysis of atherosclerotic plaques. Several methods are available that provide detailed information about vessel wall and plaque morphology . The goaIntravascular ultrasound (IVUS) is a catheter-based technology that allows for assessment of vessel wall thickness and structure. Coronary angioscopy also allows to visualize the vessel lumen. But, because of their invasive nature, these techniques are not suitable as screening methods. More recently, optical coherence tomography (OCT) and virtual histology/IVUS have been introduced as other invasive techniques that provide images of vessel wall morphology and plaque characteristics. In contrast to these invasive approaches, noninvasive technologies such as electron beam computed tomography (EBCT) allow for high resolution assessment of coronary artery luminal morphology. This method is characterized by a high negative predictive value for exclusion of coronary disease –9. Some Biomarkers are generally considered to be systemic measurements of molecules, proteins, or enzymes that provide independent diagnostic or prognostic value by reflecting an underlying disease state or condition. The clinical utility of a biomarker depends on its ability to identify high-risk patients, be accurate and reliable, and provide good sensitivity, specificity and predictive value. Clinical application further requires the demonstration that evaluation of the biomarker is not only predictive of disease, but also adds predictive value to traditional risk factors and global vascular risk assessment, such as the Framingham score. In the case of coronary artery disease, the marker must reflect the underlying biology of the vessel wall, quantifying cardiovascular specific inflammation, thereby predicting the risk of recurrent atherothrombosis and its sequelae , 13. HisCytokines are pleiotropic proteins that regulate leukocyte activity. Interleukin-6, monocyte chemoattractant protein-1(MCP-1) and TNF-α have shown promise in the prediction of coronary heart disease . PatientElevated CRP has been associated with undiagnosed peripheral, coronary, and cerebral artery disease; it differentiates patients with unstable versus stable angina; predicts future MI, stroke and sudden cardiac death in patients with coronary artery disease; correlates with MI; and predicts the presence, degree, and symptomatology of carotid stenosis, as well as early morbidity and late mortality following coronary artery bypass grafting, and late restenosis following percutaneous cardiac interventions. A major advantage of CRP over the other inflammatory biomarkers is evidence that independently adds predictive power to both lipid screening and the Framingham risk score –21. FurtIn addition to its function as a component of the generalized hepatic response to acute inflammation, CRP also appears to be produced locally in atherosclerotic plaques by resident macrophages and vascular smooth muscle cells, and may be involved in several important steps in plaque genesis, progression and rupture. CRP upregulates endothelial cell permeability, promotes endothelial adhesion, is present in subintimal plaque, fixes complement, recruits monocytes and macrophages to foci of endovascular inflammation, and stimulates local thrombogenesis. CRP may also be involved in foam cell generation, as it binds oxidized LDL with high affinity, and the resultant CRP-LDL aggregates are taken up by macrophages , 26. A nFibrinogen may increase cardiovascular risk because of its role in fibrin formation, platelet aggregation and plasma viscosity, and is also an acute-phase reactant that is elevated in inflammatory states. Levels of fibrinogen may also be useful in the identification of cardiovascular risk patients . The rolPlasma derived soluble forms of the immunoglobulin family members intercellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule-1 (sVCAM-1) have been examined as possible biomarkers in ACS . On the Oxidative stress leading to modification of LDL is an important mechanism of atherogenesis and plaque destabilization. Recent results suggest that markers of oxidative stress may have prognostic significance in ACS .Myeloperoxidase (MPO) is produced by neutrophils and monocytes at sites of inflammation. MPO can generate several reactive, oxidatively modified intermediates able to induce oxidative damage to cells and tissues. Accumulating evidence suggests that MPO may play a role in plaque vulnerability. In a prospective study, patients with ACS and elevated MPO levels had a statistically significant increase in the risk of death or MI during the first 72 hours, independently of troponin or CRP, suggesting that MPO provides independent prognostic information distinct from other established biomarkers. Advanced human atherosclerotic plaques from patients with sudden cardiac death, strongly expressed MPO at sites of rupture. The most important use of MPO may be early risk stratification of patients with NSTEMI .Oxidized-LDL (Ox-LDL) is generated during lipid peroxidation, resulting in generation of reactive species that modify the lipid components of LDL. Ox-LDL leads to foam cell formation and elaboration of pro-inflammatory cytokines that promote endothelial dysfunction. Currently three major Ox-LDL plasma ELISAs based on murine monoclonal antibodies have been developed. Circulating levels of Ox-LDL have been associated with the presence of coronary artery disease in patients undergoing elective angiography; similarly elevated Ox-LDL levels are significantly higher in patients with ACS, finally, some but not all prospective studies have shown higher Ox-LDL levels in patients subsequent development of cardiac death, non-fatal MI, and unstable angina. Therefore, Ox-LDL is an attractive biomarker, as it may provide a link between lipoprotein disorders and inflammation , 38.2 (Lp-PLA2) is a calcium-independent serine lipase that is associated with LDL in human plasma and serum. It is produced by macrophages and its expression is increased in atherosclerotic lesions , independently of a variety of potential risk factors, including markers of inflammation, renal dysfunction and hemodynamic stress  levels are significantly increased in the coronary circulation in patients with acute MI and unstable angina . CirculaPregnancy-associated plasma protein A (PAPP-A) was originally described as e peptide specifically elevated in pregnancy. Ruptured plaques have demonstrated PAPP-A expression in their shoulder. Some studies suggest that this peptidase may have diagnostic utility in identifying patients with ACS and without troponin elevation ,50.Despite some controversial reports regarding whether MMPs inhibition may have some deleterious effect, recent experimental studies demonstrate suppression of atherosclerotic plaque progression and instability by tissue inhibitors of MMPs (TIMPs), possibly through modulation of macrophage migration and apoptosis .Tissue plasminogen activator (t-PA) is a key component of the cardiovascular fibrinolytic system. In baseline conditions, t-PA is constitutively released from endothelial cells. Upon appropriate stimulation, substantial amounts of t-PA can be rapidly released resulting in a marked increase in fibrinolysis. The activity of t-PA in plasma is regulated by specific inhibitors. Of these, plasminogen activator inhibitor-1 (PAI-1) is considered to be the main inhibitor of t-PA in the vascular compartments, Additionally, α2-antiplasmin inhibits plasmin (the main proteolytic enzyme), thereby counteracting overhelming systemic fibrinolytic activity . There hCD40L, a member of the TNF family, is expressed by all the major cellular players in atherosclerosis, namely, activated T lymphocytes, EC, SMC, and macrophages. Studies examining circulating levels of soluble CD40L (sCD40L), which is primarily derived from activated platelets, have found elevated levels in patients with unstable angina and predicted the risk of future cardiovascular events in women. A recent study examining sCD40L in ACS found higher levels associated with increased risk of death or non-fatal MI, a risk that was significantly reduced with the glycoprotein IIb/IIIa inhibitor abciximab –56. HoweThe recent completion of the Human Genome Project has provided a great opportunity to identify high-risk patients through the use of technologies that integrate the entire genome. Assessment of genetic markers might predict risk of plaque instability or response to current therapies. For example, analysis of 112 polymorphisms in 2819 patients with ACS identified 3 genes  associated with increased risk of MI . AnotherAdvances in the understanding of the pathogenesis of acute coronary syndromes have stimulated development of novel biomarkers, and expanded their role in the different spectra of their underlying pathophysiology. This multi-marker strategy consists of an array of biomarkers assessing myocardial necrosis, plaque destabilization, myocardial stress, myocardial ischemia and inflammatory processes  Figure . Those wGiven the complex pathophysiology of cardiovascular disease, it is unlikely that any single biomarker will prove able to provide a universal surrogate of atherosclerosis. New imaging technology may be limited by technical difficulty, availability and cost. Soluble biomarkers may offer the advantage of availability and lower cost, but they may not prove as sensitive as imaging modalities in the detection or assessment of disease . Table 3In summary, with a multimarker approach combining emerging and new biomarkers with imaging technology, better risk profiles may emerge to provide prognostic information. A panel of inflammatory hemodynamic and vascular damage biomarkers, as part of a multimarker strategy , may helHopefully, bioimaging, by integrating biomarkers and imaging studies, offers a new opportunity to identify not only “vulnerable plaques” but also “vulnerable patients” for targeted therapeutic interventions.


Efficacy of cancer immunotherapy with cultured tumour-infiltrating lymphocytes (TILs) depends upon infused TILs migrating into tumour-bearing tissue, in which they mediate an anti-tumour response. For TILs to enter a tumour, they must first bind to tumour endothelium, and this process depends on TILs expressing and regulating the function of relevant cell-surface receptors. We analysed the cell-surface phenotype and endothelial binding of TILs cultured from human melanoma and compared them with peripheral blood T cells and with allostimulated T cells cultured under similar conditions. Compared with peripheral blood T cells, TILs expressed high levels of five integrins, two other adhesion molecules, including the skin homing molecule CLA, and several activation markers and showed markedly enhanced integrin-mediated adhesion to a dermal microvascular endothelial cell line in vitro. Compared with the allostimulated T cells, TILs expressed higher levels of the cutaneous lymphocyte antigen (CLA), the adhesion molecule CD31 and the activation markers CD30 and CD69, but lower levels of several other adhesion and activation molecules. These phenotypic and functional properties of TILs should have complex effects on their migration in vivo. Expression of CLA, the skin homing receptor, may increase migration to melanoma (a skin cancer), whereas integrin activation may cause non-specific binding of TILs to other endothelium. Manipulation of the culture conditions in which TILs are expanded might result in a phenotype that is more conducive to selective tumour homing in vivo.


In a comparative study of pre- and postmenopausal women with benign and malignant breast disease, a number of differences were observed in circulating plasma prolactin and lipid concentrations. Plasma lipids, phospholipids, triglycerides, cholesterol and free fatty acids were all higher in blood obtained from breast cancer patients prior to surgery. HDL-Cholesterol levels were significantly lower in these patients. These differences remained when the patient groups were sub-divided according to menopausal status. Plasma prolactin concentrations were also found to be higher in cancer compared with non-cancer patients, this effect being more marked in premenopausal than in postmenopausal patients. Premenopausal patients with invasive or poorly differentiated disease had significantly higher prolactin levels than those with non-invasive disease. No correlations were found between plasma prolactin and any of the lipid fractions.


Pancreatic cancer is the 5th leading cause of cancer death in both males and females. In recent years, a wealth of gene and protein expression studies have been published broadening our understanding of pancreatic cancer biology. Due to the explosive growth in publicly available data from multiple different sources it is becoming increasingly difficult for individual researchers to integrate these into their current research programmes. The Pancreatic Expression database, a generic web-based system, is aiming to close this gap by providing the research community with an open access tool, not only to mine currently available pancreatic cancer data sets but also to include their own data in the database.Currently, the database holds 32 datasets comprising 7636 gene expression measurements extracted from 20 different published gene or protein expression studies from various pancreatic cancer types, pancreatic precursor lesions (PanINs) and chronic pancreatitis. The pancreatic data are stored in a data management system based on the BioMart technology alongside the human genome gene and protein annotations, sequence, homologue, SNP and antibody data. Interrogation of the database can be achieved through both a web-based query interface and through web services using combined criteria from pancreatic  and/or public data . Thus, our database enables connections between otherwise disparate data sources and allows relatively simple navigation between all data types and annotations..The database structure and content provides a powerful and high-speed data-mining tool for cancer research. It can be used for target discovery i.e. of biomarkers from body fluids, identification and analysis of genes associated with the progression of cancer, cross-platform meta-analysis, SNP selection for pancreatic cancer association studies, cancer gene promoter analysis as well as mining cancer ontology information. The data model is generic and can be easily extended and applied to other types of cancer. The database is available online with no restrictions for the scientific community at  Pancreatic ductal adenocarcinoma (PDAC) usually presents at an advanced stage so that surgical cure is rarely achieved and conventional chemotherapy and radiotherapy have little impact, resulting in a very low 5-year survival rate (0.5%–5%) . Thus a pancreatic specimen/cell type", "pancreatic differential expression information", "genes differentially expressed in" and "genes expressed in" modules. Our design enables uploading of any available (pancreatic) datasets that comply with the structure of the pre-defined modules. Each module contains a number of subcategories related to the module name, which are fundamental to store and retrieve user-defined sub-datasets from the database by setting filters to the specific subcategories within each module. The "pancreatic specimen/cell type" module covers categories such as normal  or bulk normal pancreas (NP), acinar cells, islet cells, stromal cells and pancreatic stellate cells), and disease specimens from both exocrine , chronic pancreatitis (CP), pancreatic adenocarcinoma (PDAC), intraductal papillary mucinous neoplasms (IPMN), mucinous cystic tumours and ampullary carcinoma) and endocrine (functioning and non-functioning tumours) origin. Moreover, pancreatic juice, plasma, urine, serum, and fine needle aspirates are included as additional options to further broaden future expansion of the database. The "pancreatic differential expression information" module provides information on direction of regulation (up- and down-regulation), fold-change, SAGE tag number and whether a gene or protein was found to be expressed only in pancreatic adenocarcinoma (PDAC) or in normal pancreas. The "genes differentially expressed in" module enables more defined selection of comparison methods such as pancreatic adenocarcinoma (PDAC) versus normal pancreas , chronic pancreatitis (CP) versus normal pancreas , chronic pancreatitis (CP) versus pancreatic adenocarcinoma (PDAC), pancreatic intraepithelial neoplasias  versus normal pancreas (ND) or microdissected normal ductal cells), etc. The "genes expressed in" module lists the genes expressed in the tissue types defined in the pancreatic specimen/cell module, irrespective of their mode of regulation . The "platform technology" module enables the selection of the technology used, such as Affymetrix arrays, cDNA arrays, Sanger human 10K cDNA arrays version 1.2.1, Sanger custom 5K1 cDNA arrays, Clontech Atlas Human Cancer cDNA Expression Array, SAGE, Agilent Human Genome CGH array, 2D PAGE, SELDI, etc. The data is stored in a data management system created using MySQL [The aim of the Pancreatic Expression database is to provide a comprehensive mining tool for large-scale genomic, transcriptomic and proteomic data sets. In order to achieve this, we designed a robust internal structure encompassing specific pre-defined modules (which can be found under the "Filters" section in the database) including "ng MySQL , a simplng MySQL . We alsong MySQL  based onThe Pancreatic Expression database currently contains 32 datasets from 20 different published sources, from 14 international laboratories encompassing 22 different platforms , Affymetrix GeneChip Human Genome U133 Array Set , 2D PAGE, cDNA arrays, SAGE, Operon oligo array version 2.0, Clontech Atlas Human Cancer cDNA Expression Array, immunohistochemistry, in situ hybridisation, Oligo array, MALDI, mass spectrometry, Sanger human 10K cDNA arrays version 1.2.1, Sanger custom 5K1 cDNA arrays, United Gene Technique Ltd, BD PowerBlot Western array and qRT-PCR) -27. ThesThe Pancreatic Expression database provides access not only to bioinformatic and biostatistic experts but also to bench researchers with a limited knowledge of bioinformatics. The database allows multiple levels of access. Firstly, access to the data is provided through a customized version of MartView, a BioMart web-based query interface based on Perl API . The intNavigation between all data types is simple and user-friendly; a variety of possible query combinations allow researchers quickly to determine the most de-regulated genes and proteins across all platforms.Using the Pancreatic Expression database, it is possible to search and retrieve genes/proteins expressed only in pancreatic cancer and not in chronic pancreatitis and then ask which of these are present in urine and/or plasma. Such a query would be a first step for the discovery of non-invasive pancreatic cancer biomarkers from body fluids Figure .Researchers interested in the genes involved in the progression of pancreatic cancer can select the corresponding information among the differential expression datasets for the various tumour stages and retrieve the genes found to be de-regulated in the progression of pancreatic cancer Figure . In the Our database also allows cross-platform meta-analysis. Scientists can investigate pancreatic expression profiling performed across a wide range of different platforms (such as cDNA arrays or oligo arrays) to detect the most consistent sets of de-regulated genes Figure . ImportaAs they are available through the BiomaRt package , annotatOur integration model brings together relevant pancreatic cancer datasets and annotations from public sources and enables scientists to perform a wide variety of complex queries on various types of data. The design of the database allows easy integration of additional modules and annotations from new public databases.The Pancreatic Expression database constitutes a unique and valuable resource for the wider cancer research community, and is in rapid and constant development. We aim to continuously import new data sources and update the database on a regular basis, and invite scientists worldwide to deposit and share their data.Although initially constructed using pancreatic cancer expression datasets, we have designed and implemented a generic system that can be easily modified and applied to any other type of cancer. The system is available for collaboration with all interested research groups either by extending it to include other cancer data or by sharing our model should they want to adopt it for their data.Project name: Pancreatic Expression databaseProject home page: Operating system(s): Platform independent; Standard WWW browser Programming language: Perl, SQL, BioMart data management systemLicence: The database is freely available to academic and non-academic users. However, should you find the Pancreatic Expression database useful to your work, please cite this paper.CC designed and implemented the web site and database, annotated and integrated the data, contributed to the data collection and wrote the manuscript. SAH defined the pancreatic cancer modules, collected pancreatic data, tested the database, contributed to the revision of the manuscript and continuous discussion. HJW, SB, DH and TPR were involved in the pancreatic data collection. TCJ contributed to the definition of the pancreatic cancer modules and pancreatic data collection. TCJ and NRL provided valuable guidance and expertise on pancreatic cancer, contributed to the critical revision of the manuscript and continuous discussion. All authors read the final manuscript.


This study considers gene location within bacteria as a function of genetic element mobility. Our emphasis is on prophage encoding of bacterial virulence factors (VFs). At least four mechanisms potentially contribute to phage encoding of bacterial VFs: (i) Enhanced gene mobility could result in greater VF gene representation within bacterial populations. We question, though, why certain genes but not others might benefit from this mobility. (ii) Epistatic interactions—between VF genes and phage genes that enhance VF utility to bacteria—could maintain phage genes via selection acting on individual, VF-expressing bacteria. However, is this mechanism sufficient to maintain the rest of phage genomes or, without gene co-regulation, even genetic linkage between phage and VF genes? (iii) Phage could amplify VFs during disease progression by carrying them to otherwise commensal bacteria colocated within the same environment. However, lytic phage kill bacteria, thus requiring assumptions of inclusive fitness within bacterial populations to explain retention of phage-mediated VF amplification for the sake of bacterial utility. Finally, (iv) phage-encoded VFs could enhance phage Darwinian fitness, particularly by acting as ecosystem-modifying agents. That is, VF-supplied nutrients could enhance phage growth by increasing the density or by improving the physiology of phage-susceptible bacteria. Alternatively, VF-mediated break down of diffusion-inhibiting spatial structure found within the multicellular bodies of host organisms could augment phage dissemination to new bacteria or to environments. Such phage-fitness enhancing mechanisms could apply particularly given VF expression within microbiologically heterogeneous environments, ie, ones where phage have some reasonable potential to acquire phage-susceptible bacteria. Viruses are either the simplest of living things or are among the more complex of non-living things. At the very least, viral gene pools can form a continuum with those of their hosts, with gene exchange and recombination occurring between viral and host DNA. This gene exchange can be so pervasive that the viruses of bacteria—known as phages or bacteriophages—have been suggested as substantial or even the most significant drivers of bacterial evolution . So-callEscherichia coli such as E. coli O157 —agents known to contribute to the development of infectious disease in eukaryotes such as ourselves—have been shown to be encoded by prophage . Human doli O157 . Indeed,oli O157  as well oli O157 .Though phage encoding of virulence-factor genes is important to an understanding of many bacterially mediated infectious diseases, here we will consider a more fundamental issue: Why do phage, particularly prophage, even encode VFs? In principle there exist at least four entities that could selectively benefit from the resulting association: the VF gene, the bacterium host, bacterial populations, and the encoding phage/prophage. Furthermore, a given VF-associated phenotype could simultaneously benefit any or all of these four entities. We list in virions of lytic phage to be distinct organisms. That is, as bacterial predators, parasites, or horizontally transmitted entities, the virions of lytic phage, even of temperate phage, exist at least semi-autonomously from their bacterial hosts. Such a perspective— which we believe may be key to understanding why temperate phage appear to commonly encode bacterial VFs—contrasts a viewing of phage  instead as somewhat integral components of various bacterial pathogens. In terms of levels of selection, we therefore order our discussion of the mechanisms contributing to phage maintenance of bacterial VF genes by going from gene to prophage to individual bacterium to bacterial population, but only subsequently, and separately, do we discuss selection acting upon phage virions. Thus, we consider a three-organism/four-component system—phage, bacterial host, eukaryotic host, and VF—where VFs are active against eukaryotic hosts, are expressed within and by bacteria, and are horizontally transmitted between bacteria by the phage virions produced by induced prophage.The order in which we discuss these mechanisms reflects our bias in considering especially the Where appropriate we extend our emphasis to include non-phage mobile genetic elements MGEs; , particumaintained.Selection acting on individual bacteria does not necessarily favor a continued gene-prophage or, more generally, a gene-MGE association. For instance, if a gene confers some benefit to a harboring bacterium, but an encoding prophage does not otherwise confer any advantage to the same bacterium, then over time we would expect evolution to favor a deletion of the prophage sequences accompanying a given gene  see Ber. Alternabetter than the equivalent gene already found in that bacterium, a scenario that is unlikely given a lack of a priori coevolution between gene and bacterium, or alternatively (and more likely) provide or augment a novel or existing function  acquisition. By contrast, there exists a second gene-centered explanation for maintenance of gene-MGE associations—one that presumes differences in bacterial fitness prior to, rather than following, prophage or MGE acquisition (The above-posited mechanism(s) suggest that some combination of gene mobility and infrequent selection could be sufficient to maintain gene-prophage (or gene-MGE) associations. Note that we made no assumption of fitness differences between bacteria receiving MGE-associated genes, except uisition . Here thuisition , should Still, from the bacterium’s perspective, once genes have been acquired there should be little incentive to retain, via this hitchhiking mechanism, a gene-prophage  association : Not onlIf greater bacterial invasiveness results in an increase in bacterial susceptibility to a host’s immune response, and acquisition of an MGE-encoded VF gives rise to greater bacterial invasiveness eg, , then VFIf the bacterium’s antigenicity is bacterium encoded rather than bestowed by a MGE-encoded gene, then MGE association would allow the VF gene to relatively easily acquire alternative bacterial hosts that are not currently recognized by an animal’s immune response. To our knowledge this specific scenario has not been observed within a single animal over the course of bacterial infection. Over a community of animal hosts, however, such a scenario would be equivalent to VF horizontal transfer to bacteria displaying a wide range of antigenic variation  and woulunassociated with phage genomes. One possibility is that it is those genes that are less-often employed by bacteria  that are less readily retained within the bacterial genomes by natural selection. To avoid extinction, such less-employed genes presumably can more readily benefit from, and indeed may require, some combination of MGE-mediated population expansion and virion-mediated longer-term survival. These explanations, however, return us to the concept of peace offers and the question of whether or to what extent intermittent or non-essential gene utility to bacteria can select for ongoing VF association with prophage.Phage-mediated mobility can result in faster gene evolution through increased rates of mutation, recombination, or adaptation . ImpliciThe first of these enhanced gene-evolution hypotheses is presented by context of gene expression and evolution.Genes associated with MGEs may also experience more recombination events with related genes than do chromosome-bound genes . For exaprophage at least potentially expendable, or the carried gene is not often useful to the phage or the bacterium, thereby rendering the gene expendable. Consistent with the Selfish Operon Model of While mobility by itself may confer a selective advantage on a gene, such enhancement of a gene’s fitness does not necessarily select for gene retention within a phage genome. Either a prophage-carried gene is useful to a prophage-infected bacterium, with the There are at least three mechanisms that could, by enhancing bacterial propagation, contribute to maintenance of an association between VF genes and prophage: (i) expression of phage genes leading to an increase VF utility to bacteria, (ii) virion packaging of VF genes to promote community-wide mechanisms of bacterial pathogenesis , and (iii) phage release associated with VF expression that results in a numerical reduction in bacteria capable of directly competing with the unlysed kin of induced lysogens . These are mechanisms 6, 7, and 8 as presented in Corynebacterium diphtheriae, for example, can be enhanced by the induction of the encoding prophage  of gene with MGE . Diphtheprophage . Reliancironment . Lendingironment . Furtherromosome . Also coIn this section we consider how advantages associated with VF mobility might stem from disseminated expression of a VF gene. An assumption is made that the advantages associated with the expression of certain VFs  may be shared among bacteria and furthermore that these bacteria may benefit from the collective production of a greater VF dose. To achieve this greater VF production it may be possible to disseminate the VF genes—via MGEs such as phages—throughout communities of normal-flora bacteria. This effect may be described as the dissemination of an effective VF (or toxin) dose . From BiC. diphteriae the bacteria double every hour in vitro, but in that same hour a lytic corynephage can produce 30–60 converting phages. Nontoxigenic C. diphtheriae, S. pyogenes, S. aureus, and E. coli are all frequent if not constant members of the normal human flora. For these autochthonous organisms, in situ phage conversion represents a means of rapid spread of the toxin gene once a susceptible host has been infected with just a few toxinogenic organisms.Phage conversion is a highly efficient means of rapidly disseminating the toxin gene within a nonimmune host. In the case of in situ. Such interactions between phage and various bacterial pathogens can indeed occur over the course of at least experimental infection of animal hosts  and also is potentially the case for two prophage-encoded Streptococcus mitis platelet-binding factors  or, more generally, can involve either lysogeny, chronic phage infection, or, alternatively, the lytic infection of some though not all bacteria in a population—all of which may be described as phage conversions . For lysin situ characterization of (i) probabilities of lytic-lysogenic decisions, (ii) the potential of purely lytic infections to produce phage-encoded toxins, (iii) phage host range, and (iv) whether inclusive-fitness benefits really are sufficient to select for VF retention among bacterial pathogens. Some of these considerations have been addressed for V. cholerae, which potentially employs phage-mediated toxin dose dissemination in the course of infection  lysogens may accrue any benefit associated with toxin production just as potentially competing bacteria  are infected and then lysed by these phage.Inefficient reduction to lysogeny can provide an alternative explanation of why toxin genes may associate with prophage, via a phenomenon that Salmonella . In this section we reflect on the VF impact on phage propagation. We consider a positive impact to be something that results in an increased phage burst size, a decreased phage latent period  or in terms of successful phage dissemination to new ecosystems (between-culture phage transmission). Phage fitness therefore may be enhanced if exotoxins are capable of (i) increasing the health or metabolic activity of phage-susceptible bacteria  advantage if prophage are defective, which in turn collapses to assumptions only of inclusive fitness if the host bacterium also must be killed to effect exotoxin expression. In the first instance the prophage cannot take advantage of improvement in conditions for virion-mediated population growth because the prophage cannot produce functional virions. In the second instance, even the expressing bacterium cannot take advantage of improvements in conditions because the bacterium dies long before conditions improve. Note, then, the narrow distinction between inclusive fitness gains and phage gains: Inclusive-fitness gains are a function solely of the fitness gains made by intact bacteria that also harbor the toxin gene whereas non-defective prophage, by releasing free-phage progeny, additionally may benefit from gains made by phage-susceptible bacteria—bacteria that do not necessarily harbor the toxin gene. Thus, there is a similarity between these seemingly distinct gains, bacterium versus phage: Both, as posited, have to do with ecosystem modification such that bacteria, at least over the short term, display an enhanced potential for growth.This view of phage modifying their environment in a manner that enhances bacterial growth is not necessarily limited to the impact of phage-encoded VFs. Indeed, phage-induced bacterial lysis alone should result in the release of previously sequestered nutrients, ie, those formerly associated with not-lysed bacteria, which could then become available to surviving bacteria .Increasing physical movement, like increasing nutrient densities, could also benefit phages, resulting in selection for continued association between phage and movement-enhancing VF genes. Such movement could occur within as well as between bacteria-containing environments. Within bacteria-containing environments various phage-mediated modifications of virion movement are already known, particularly ones that facilitate phage movement to the surface of phage-susceptible bacteria. Enzymatic degradation of materials associated with the bacterium that a phage is adsorbing—with enzymes displayed, for example, by virion particles— are probably fairly common  dissemination out of animals is enhanced, phage-encoded enterotoxins might still augment the fitness of encoding phage by means of local enhancement of virion dissemination.Diarrhea-like loosening of stools might also foster more-rapid mixing and phage diffusion within environments, thereby resulting in greater availability to phage of phage-susceptible bacteria within colons. Diarrhea, therefore, may be described as potentially aiding free-phage transmission to new bacteria Perhaps the most striking conclusion from our analysis of mechanisms potentially retaining phage-VF gene associations is the relative dearth of mechanisms enhancing the fitness of individual bacteria. The sole mechanism we have identified is epistatic interactions between phage and VF genes (mechanism 6), but only given that linkage between those genes is a reasonable response to such epistasis and even then only if VF expression doesn’t coincide with death of the bacterial lysogens . Instead, the bottom line for phage-VF gene association is that for such an association to be stably maintained there must be an advantage for the gene to remain in association with a functional phage genome. For a phage genome to be functional, from an ecological vantage, then phage-susceptible bacteria must be obtainable by released phage progeny such that the phage life cycle may be completed. We would argue that this functional requirement for bacteria to infect, and not just for phage production, could significantly constrain the evolutionary maintenance of associations between functional phage genomes and VF genes. That is, bacterial infections that are too invasive may be too homogeneous in terms of bacterial types present to provide temperate phages with susceptible bacterial prey, ie, ones not already lysogenized by the same phage. Too-invasive bacterial infections also may be too sealed off from the outside world to provide phages with a reasonable potential to disseminate to susceptible bacteria found elsewhere, other than via the normal bacterial portal of exit.Corynebacterium diphtheriae, Shiga toxin as produced by Shiga toxigenic E. coli such as E. coli O157, and Cholera toxin as produced by Vibrio cholerae—are all associated with infections in which bacteria do not actively invade host tissues but instead are limited to colonization on mucous membranes (eg, For these more sealed off infections we would envisage a closer correspondence between phage and bacterial interests, including mechanisms of dissemination. That is, there could be less reason for eukaryote-tissue modifying genes to be phage rather than bacterium encoded. Mucous membranes, by contrast, more likely carry normal-flora bacteria than do tissues that are deeply buried within animal bodies. It is of interest in terms of VF utility, therefore, that the three best studied of phage-encoded exotoxins—diphtheria toxin as produced by anes eg, .in situ such as rates of lysogen induction, relative likelihoods of lytic versus lysogenic infection, phage burst size, phage host range, evolutionary dynamics of phage-associated genes, and the ability of purely lytic infections to produce bacterial toxins. Ultimately this knowledge of phage biology may promote not just a better understanding of those forces impacting phage-VF associations, but also a better understanding of prophage-mediated disease processes themselves.We suggest that the phage utility of VFs in general and the phage utility of exotoxins in particular may be found under those circumstances where modification of the eukaryote-host environment allows increased phage potential for acquisition of new bacterial prey. In these circumstances phage biology and ecology—eg, as by disseminating an effective toxin dose—may impact dramatically on resulting disease. To better understand not only the evolution of bacterial pathogens, but also the progression of bacterial disease, it therefore may be necessary to understand phage-encoded VFs not just from the perspective of expressing bacteria, but also in terms of the ecology of entire ecosystems. Of great pertinence, therefore, is determining which factors control phage impact


Previous studies have demonstrated steep gradients of decreasing doxorubicin fluorescence with increasing distance from blood vessels, such that many tumor cells are not exposed to drug. Studies using multilayered cell cultures show that increased P-glycoprotein (PgP) is associated with better penetration of doxorubicin, while PgP inhibitors decrease drug penetration in tumor tissue. Here we evaluate the effect of PgP expression on doxorubicin distribution Mice bearing tumor sublines with either high or low expression of PgP were treated with doxorubicin, with or without pre-treatment with the PgP inhibitors verapamil or PSC 833. The distribution of doxorubicin in relation to tumor blood vessels was quantified using immunofluorescence.Our results indicate greater uptake of doxorubicin by cells near blood vessels in wild type as compared to PgP-overexpressing tumors, and pre-treatment with verapamil or PSC 833 increased uptake in PgP-overexpressing tumors. However, there were steeper gradients of decreasing doxorubicin fluorescence in wild-type tumors compared to PgP overexpressing tumors, and treatment of PgP overexpressing tumors with PgP inhibitors led to steeper gradients and greater heterogeneity in the distribution of doxorubicin.PgP inhibitors increase uptake of doxorubicin in cells close to blood vessels, have little effect on drug uptake into cells at intermediate distances, and might have a paradoxical effect to decrease doxorubicin uptake into distal cells. This effect probably contributes to the limited success of PgP inhibitors in clinical trials. Effective systemic treatment of solid tumors requires that constituent cells be sensitive to the drug(s) used and that the drug(s) be able to achieve a concentration within the cells sufficient to cause cytotoxicity. The ability of anti-cancer drugs to gain access to all of the viable cells in solid tumors depends on efficient delivery of drugs through the vascular system, and on penetration of drugs to tumor cells that are distant from blood vessels. Solid tumors have an inefficient bloody supply with tortuous and leaky vessels, large intercapillary distances and intermittent blood flow. There is evidence that several anticancer drugs have poor penetration of tumor tissue from blood vessels -5. In pain vivo.Many of the anticancer drugs in clinical use are natural products, or derivatives of natural products  and they share common mechanisms of resistance. Many of these drugs are high affinity substrates for energy-dependent membrane transporter proteins that act to pump drugs out of cells. The best characterized of these drug efflux pumps is P-glycoprotein (PgP) -10. P-glex vivo tumor tissue models containing cells expressing low levels of PgP and those with high PgP expression, and these distributions can be modified by PgP inhibitors  doxorubicin through drug-resistant MCCs (PgP overexpressing) compared to drug-sensitive MCCs (wild-type) [Drug distribution may be influenced in tumor tissue by the presence of high interstitial fluid pressure, an extensive extracellular matrix, cell-cell contact and the expression of efflux pumps such as PgP on the cell surface. It has been proposed that since PgP and other drug transporters reduce cellular accumulation of their substrates, they might assist in transporting the drugs to neighboring cells farther away from blood vessels, resulting in a net increase in tissue penetration of chemotherapeutic agents . This hyld-type) ; inhibitld-type) . While uld-type) ,31. Whenld-type) . MulticeHere, we have investigated the effects of PgP overexpression and PgP inhibitors on doxorubicin distribution in solid tumors grown in mice. The distribution of doxorubicin is quite variable within tumor tissue. In well-vascularized areas drug distribution is relatively uniform; however in most areas of the tumor where there are few blood vessels, distribution to cells distal from blood vessels is limited so that many viable tumor cells have minimal exposure to drug, allowing them to survive and proliferate. For this reason, we selected areas of interest within a tumor section that had low vascular density. We examined doxorubicin distribution 10 minutes after intravenous administration because previous studies in our laboratory have shown no difference in doxorubicin distribution in relation to the nearest blood vessel beyond this time point .Similar to data for MCCs, our results show an increase in doxorubicin distribution in the PgP overexpressing tumors AR1 and MCF-7/ADR compared to their wild-type variants EMT6 and MCF-7, respectively. There is rapid cellular uptake of doxorubicin in wild-type cells and this is largely due to its ability to bind tightly to DNA , and in Methods of inhibiting PgP have been studied extensively for over two decades. Many agents that modulate PgP transport such as verapamil and cyclosporin were identified in the 1980s, and were evaluated as chemosensitizing agents. These agents produced disappointing results in clinical trials in part because their low binding affinities necessitated the use of high doses, resulting in unacceptable toxicities . In our Second generation PgP modulators include valspodar (PSC 833), biricodar, and dexniguldipine. These agents are more potent and specific than first generation inhibitors . ValspodAlthough cytotoxicity and pharmacokinetic interactions explain partially why PgP inhibitors have not been very effective in clinical trials, a clear explanation for the minimal effects to increase mean cellular accumulation of drug has yet to be provided. Studies to determine cellular accumulation often involve homogenizing tumor tissue and conducting high-performance liquid chromatography (HPLC) ,37. ThisOur study uses a non-orthotopic tumor mouse model to show that tumor cells in areas far from blood vessels where PgP inhibitors have no effect (or possibly even a negative effect) due to changes in distribution of doxorubicin, might counterbalance areas close to blood vessels where uptake of doxorubicin into tumor cells is increased, thus limiting the effectiveness of these inhibitors. While tumor dynamics, vasculature and heterogeneity affect drug distribution, this non-orthotopic mouse model is still limited in fully representing the conditions in human patients.Computer- simulated mathematical models have shown that adequate drug distribution is a crucial factor in determining drug effectiveness . One moddecreased drug uptake in more distal cells (Figure We have shown that PgP expression and inhibitors of PgP function can influence doxorubicin distribution in some solid tumors. The distribution of doxorubicin is more heterogeneous in wild-type tumors, than in those where constituent cells express PgP: drug uptake is higher in cells close to blood vessels in wild-type tumors, but there are minimal differences in drug uptake by more distal cells. Both the first-generation inhibitor verapamil and the second-generation inhibitor valspodar alter drug penetration in both a murine tumor and a human xenograft, leading to improved uptake of doxorubicin only in tumor cells within a restricted radius around functional blood vessels. Whether PgP overexpressing tumors are treated with a PgP inhibitor or not, cells distal to blood vessels at approximately 50-120 μm, show no significant difference in doxorubicin fluorescence (Table s Figure . These rThe authors declare that they have no competing interests.in vivo drug distribution studies, imaging, analysis and growth delay studies as well as drafting and revising the manuscript. IFT conceived the concepts underlying the study, designed the experiments, revised the manuscript and provided funding, grant support and overight.KJP participated in designing, planning and carrying out The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/356/prepub


The past is never dead. It's not even pastWilliam Faulkner (1951)Bacteria can acquire heritable immunity to viral (phage) enemies by incorporating phage DNA into their own genome. This mechanism of anti-viral defence, known by the acronym CRISPR, simultaneously stores detailed information about current and past enemies and the evolved resistance to them. As a high-resolution genetic marker that is intimately tied with the host–pathogen interaction, the CRISPR system offers a unique, and relatively untapped, opportunity to study epidemiological and coevolutionary dynamics in microbial communities that were previously neglected because they could not be cultured in the laboratory. We briefly review the molecular mechanisms of CRISPR-mediated host–pathogen resistance, before assessing their potential importance for coevolution in nature, and their utility as a means of studying coevolutionary dynamics through metagenomics and laboratory experimentation. It is therefore convenient when evolution leaves behind traces of its activity. Fossils record the past, but offer a poor record of evolution at the molecular level. Fortunately, evidence of the past is often imprinted upon DNA sequences. For example, sequence comparisons can be used to estimate the proportion of differences between species that are driven by natural selection , and even to estimate the speed at which adaptation has proceeded . Such an2.Viruses that infect bacteria (bacteriophages) are ubiquitous in natural bacterial communities , and in An integral part of this anti-phage defence mechanism is the action of CRISPR-associated (Cas) proteins. Cas proteins are usually found adjacent and upstream of CRISPR arrays, and show high sequence and structural similarity to proteins with endonuclease, and DNA- and RNA-binding functions a, which The molecular details are still being unravelled, but the anti-viral action of the CRISPR–Cas complex appears to proceed in three distinct stages a. In theet al. http://crispr.u-psud.fr and http://crispi.genouest.org). These ever-growing interactive databases find CRISPR–Cas complexes in roughly 40 per cent of bacterial and nearly 90 per cent of archaeal genomes tested so far. Up to 45 Cas protein families have been described based on amino acid sequences , analysis of CRISPR arrays gave strikingly different results from a thorough analysis of the rest of the bacterial genome. Specifically, analysis of CRISPR arrays revealed that there were no common spacer sequences between the two populations, while there was very little divergence at 48 other loci. To reconcile these differences, the authors hypothesized that CAP strains disperse widely to become genetically homogeneous (hence to very low divergence found in most of the genome), but differences in exposure to local phage populations lead to rapid divergence of CRISPR loci. By sequencing the phage meta-genome from the USA bioreactor, they found 11 CRISPR spacers with matches to sympatric phage genome fragments. Thus, the CRISPR record of interaction with phage, coupled with geographical sampling, suggests a coevolutionary mosaic that overpowers the effects of dispersal. A similar result was found in a comparative genomic analysis of Sulfolobus islandicus CRISPR arrays and spindle-shaped virus (SSV) sequences, where viruses exhibited clear biogeographic structure apparently driven by ongoing adaptation to local host strains  or acidophilic (Leptospirillum) bacteria and their natural phage pathogens (Leptospirillum populations found in acid mine drainage and subaerial biofilms (39°C, pH approx. 1; CRISPR studies have shown that our inability to culture the vast majority of bacteria does not necessarily stop us from accurately inferring coevolutionary dynamics. This is certainly the case for microbial biofilms of thermophilic (athogens  and an aPseudomonas bacteria and phage (Daphnia and long-living parasite transmission spores from dated lake sediment cores (The studies described above used a spatial sampling strategy to test for local adaptation. Such spatial patterns of local adaptation can indirectly indicate the action of coevolution, but regular and frequent sampling through time offers a more direct window on the process. As with spatial sampling, it would add value to confirm field patterns of phage adaptation with laboratory experimentation, specifically via time-shift experiments, where antagonists are sampled at different time points and controlled infections carried out between them . By obsent cores  reportednt cores .Work in some host–pathogen systems has sought to infer the nature of coevolutionary dynamics from the underlying genetics of infection. Most notably, workers have compared the propensity of the ‘gene-for-gene’ and ‘matching allele’ infection models to promote and maintain genetic polymorphism . HoweverOne may also speculate on the relative importance of CRISPR-based resistance relative to other phage-resistance mechanisms, such as cell-surface receptor modification. Receptor modification offers a first line of defence that impedes the entry of most phages, but mutations at these receptors often come at some metabolic cost to the host, as they are the same receptors involved in nutrient uptake (e.g. 4.Coevolutionary dynamics and levels of polymorphism in resistance are tightly linked with the costs associated with resisting infection . Costs oThere are several potential ways that CRISPRs might be a costly system. As hosts incorporate more spacers , longer 5.Evolutionary biology is, in principle, well poised to understand the processes and mechanisms that underpin changing pathogenicity, with well-established general theories of adaptation , invasioWhile the existence of CRISPR arrays in prokaryotes was originally recognized in the late 1980s (