Obesity and its related illnesses are pan-endemic health problems which require intervention. Laparoscopic Adjustable Gastric Banding (LAGB) is seen as a safe surgical procedure with satisfactory results on weight reduction and improvement in obesity related illness.Data were collected in a repeated-measures longitudinal five year study for 143 morbidly obese patients who underwent laparoscopic adjustable gastric banding (LAGB). Follow up was continued from 3 to 60 months post operatively. Patients were assessed for diabetes, hypertension and dyslipidemia.Repeated measures ANOVAs revealed that both men and women lose weight and reduce their BMIs at a consistent rate. At 3, 6 and 9 months post-operative there are no differences in percent weight loss between men and women with mean weight loss at 8.9%, 13.1% and 16.0% respectively of pre-operative weight. At 12, 24 and 60 months post-operatively, however, men significantly increase the percentage of weight loss as well as improve their BMI compared to women (p < .05). At 24 months post-operative, men and women have BMIs of 36.03 and 32.85, both still in the obese range. By 60 months men have achieved a BMI that is slightly under the obese range into the overweight range (30.76) while women (BMI = 36.61) were still in the obese range. At 60 months, men have lost a total of 33.75% of their pre-operative body weight while women have lost a total of 21.50. Diabetes, hypertension and dyslipidemia were significantly reduced in the sample post-operatively (p < .01).LAGB is a safe and effective surgical procedure for morbidly obese patients resulting in weight loss, BMI decrease and reduction in co-morbid illnesses. The mean body mass index (BMI) increased dramatically from 24 kg/mThe WHO has recognized the impact of obesity on health and quality of life and thus recommended that studies of health changes associated with weight loss should be a research priority  and laparoscopic Roux-en Y gastric bypass (LRYGBP). LRYGBP is the most common bariatric surgery in the USA whereas in Europe and Australia, LAGB is more common  underwent LAGB between June 2003 and February 2010. Patient selection was based on the criteria that has been identified by the The mean weight pre-operative for women was 118.74 kg with BMI = 45.75 and for men was 137.51 kg with BMI = 45.30. Patients were assessed for co-morbidities, operative time, operative complication, hospital stay, and follow up at 3, 6, 9, 12, 24 and 60 months. The mean operative time was 70.2 minutes (SD = 23.5). There were no major complications or patient mortality during the study period. All patients had undergone various conservative approaches  for morbid obesity over a period of twelve or more months without any effect.Surgical technique - All of the LAGB surgeries were performed by a single gastro-intestinal (GI) surgeon (KA) in one hospital . The procedure involved insertion of four to five trocars in the upper abdomen. The band (Swiss adjustable band) was placed around the fundus of the stomach straight away below the esophagogastric junction via a retrogastric tunnel created using the pars flaccid approach. In some cases, the band was fixed by two or three anterior imprecating gastro-gastric sutures.Postoperative first visit - The initial follow up visit was at one week postoperatively. Reviews continued monthly for the first year, and then reduced to a visit every two months in the following years. All adjustments to the band were performed with 7–8 ml of contrast under fluoroscopic imaging.Co-morbid disease - Patients with the following co-morbidity  were identified. For each patient suffering from diabetes, fasting blood sugar and Hba1c was recorded. Patients with dyslipidemia, cholesterol level and low density lipoprotein (LDL) were measured. All hypertensive patients were evaluated according to their blood pressure measurement and medication dosage pre and postoperatively. Co-morbid illness was considered resolved if the patient no longer required medication after surgery and specific laboratory values returned to normal. Improvement of co-morbidity was defined by dose reduction in medication with improvement in specific laboratory values. Treatment of co-morbid conditions was supervised by the relevant specialist.Operative complication - Intra-operative and post operative complications were recorded. Complications in the form of intra-operative bleeding, band intolerance, and wound infection were recorded. No gastric perforation, necrosis, or band slippages were detected.Data analyses were conducted with SPSS version 19.0. In addition to descriptive statistics, a two-way repeated measures analysis of variance (2-way rep-ANOVA) employing sex (2 levels) and time (7 levels) as independent variables and BMI as the dependent variable was conducted. A second 2-way rep-ANOVA employing sex (2 levels) and time (7 levels) as independent variables and weight (kg) as the dependent variable was conducted. Changes in co-morbidities were analyzed with Fisher’s exact test. The level of significance was set at p < 0.05.The study was approved by the Ethics Committee of Bahrain Defense Force Hospital. Each patient provided informed consent to take part in the study.There was significant differences in the first 2-way rep-ANOVA (sex × time) with BMI as the dependent variable  as there was for weight (kg) as the dependent variable . These results are summarized in Table Additionally, the change in BMI from pre-operative to post-operative periods is represented graphically in Figure Analyses of the co-morbidities (at 24 months) indicated that the majority of diabetics pre-operative (n=18) were non-diabetic post-operative . Similarly, 19 patients had hyperlipidemia pre-operatively but this number was reduced to 6 post-operatively . Finally, 14 patients had hypertension pre-operatively and this was reduced to 5 patients post-operatively . There was, therefore, a significant reduction in all three co-morbidities that were assessed.This is the first study with a five year follow-up of LAGB in Bahrain. It confirms that LAGB is a safe and effective surgery with a good result in weight reduction and changes in co-morbid illnesses. It also confirms that LAGB can be performed with a minimum of significant complications, which has been confirmed by other studies (1991).Weight reduction is the most commonly reported outcome measure of bariatric surgery  - cholesterol concentration and increased low-density lipoprotein (LDL) is highly atherogenic and the most common pattern associated with coronary disease (Bacci et al. LAGB is an effective and safe procedure for weight loss in morbidly obese patients. After gastric banding, patients with morbid obesity experience a significant reduction in major co-morbid illness. The benefit of sustained significant weight reduction following LAGB surgery on the serious side-effects of obesity was demonstrated by its impact on diabetes, hypertension and dyslipidemia. Therefore, the success of LAGB should be judged by the weight loss and the improvement of obesity-associated co-morbidities.Notwithstanding the positive results, further research is required to explore sex differences in the results of LAGB, as well as other cross-cultural differences that may exist. Moreover, continued further longitudinal studies of LAGB will be useful to further evaluate the long term impact of this bariatric procedure. Meanwhile, the results of the present study indicate that LAGB is a safe and effective surgical procedure for morbidly obese patients resulting in weight loss, BMI decrease and reduction in co-morbid illnesses.


Viral and bacterial respiratory tract infections in early-life are linked to the development of allergic airway inflammation and asthma. However, the mechanisms involved are not well understood. We have previously shown that neonatal and infant, but not adult, chlamydial lung infections in mice permanently alter inflammatory phenotype and physiology to increase the severity of allergic airway disease by increasing lung interleukin (IL)-13 expression, mucus hyper-secretion and airway hyper-responsiveness. This occurred through different mechanisms with infection at different ages. Neonatal infection suppressed inflammatory responses but enhanced systemic dendritic cell:T-cell IL-13 release and induced permanent alterations in lung structure . Infant infection enhanced inflammatory responses but had no effect on lung structure. Here we investigated the role of hematopoietic cells in these processes using bone marrow chimera studies.C. muridarum. Nine weeks after infection bone marrow was collected and transferred into recipient age-matched irradiated naïve mice. Allergic airway disease was induced (8 weeks after adoptive transfer) by sensitization and challenge with ovalbumin. Reconstitution of irradiated naïve mice with bone marrow from mice infected as neonates resulted in the suppression of the hallmark features of allergic airway disease including mucus hyper-secretion and airway hyper-responsiveness, which was associated with decreased IL-13 levels in the lung. In stark contrast, reconstitution with bone marrow from mice infected as infants increased the severity of allergic airway disease by increasing T helper type-2 cell cytokine release (IL-5 and IL-13), mucus hyper-secretion, airway hyper-responsiveness and IL-13 levels in the lung. Reconstitution with bone marrow from infected adult mice had no effects.Neonatal (<24-hours-old), infant (3-weeks-old) and adult (6-weeks-old) mice were infected with These results suggest that an infant chlamydial lung infection results in long lasting alterations in hematopoietic cells that increases the severity of allergic airway disease in later-life. Chlamydia and Mycoplasma) infections in early-life are thought to contribute to the development of asthma Asthma is a chronic inflammatory disease of the airways that is particularly common in children. The clinical course of asthma is punctuated by recurring exacerbations that are underpinned by aberrant CD4+ T helper type 2 lymphocyte (Th2 cell) responses to environmental stimuli Chlamydia pneumoniae is a notable exception and is increasingly linked with the development of asthma in both children and adults The etiology of asthma suggests that altered immunological programming in early-life by specific infections may play critical roles in the induction and progression of the disease. There is emerging evidence to suggest that both the nature and timing of infection is pivotal in determining whether specific infections protect or predispose to asthma. Numerous studies show an inverse association between Th1-inducing infections and the development of asthma C. pneumoniae are common and usually asymptomatic but are responsible for up to 22% of all cases of community-acquired pneumonia requiring hospitalization C. pneumoniae antibodies Chlamydia can infect dendritic cells (DCs) and subvert their function to induce Th2 responses and AHR Respiratory infections with Recent studies suggest that hematopoietic cells can respond directly to infections and inflammatory signals C. muridarum as infants, but not neonates, increases the severity of AAD in later-life. Therefore, early-life infection-induced alterations in hematopoietic cells may play a previously unrecognised role in predisposing to severe AAD.The effects of chlamydial lung infection on hematopoietic cell function and subsequent AAD have not been investigated. In this study, we demonstrate that reconstitution of bone marrow from mice infected with All experiments were performed with approval from the animal ethics committees of The University of Newcastle and Garvan Institute/St. Vincent's Hospital, NSW.Specific pathogen-free pregnant and non-pregnant BALB/c mice  were obtained from the central animal house, The University of Newcastle or from Australian BioResources .C. muridarum  or 30 µl (infant and adult) sucrose phosphate glutamate buffer [vehicle]) Neonatal (<24 hour old), infant (3 weeks old) or adult (6 weeks old) BALB/c mice were infected intranasally with 7 cells were intravenously transferred to recipient age-matched irradiated naïve BALB/c mice. Recipient mice were irradiated twice (four hours between each irradiation) with 450RAD (4.5 Gy) prior to adoptive transfer of bone marrow Nine weeks after neonatal, infant or adult infection, or sham inoculation, bone marrow was extracted from the hind limbs of donor mice and 1×106 MLN cells plated out in 96 well culture plates in 200 µl of RPMI 1640 culture medium containing 10% FCS, 2 mM L-glutamine, 2 mM sodium pyruvate, 100 µg/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-mercaptoethanol . Cells were re-stimulated with Ova  for 4 days and the concentration of IL-5, IL-13 and IFN-γ in supernatants were analyzed by ELISA using paired antibodies  as we have previously described Mediastinal lymph node (MLN) cells were excised, single cell suspensions prepared, erythrocytes lysed and 1×10Lungs were formalin-fixed, embedded, and sectioned (4–6 µm). Sections were stained with periodic acid–schiff for enumeration of MSCs in the airways as we have previously described Lung function, in terms of AHR, was measured in anesthetized mice using whole-body plethysmography by determination of transpulmonary resistance and dynamic compliance in response to increasing doses of nebulized methacholine (Sigma) as we have previously described Whole lungs were homogenized in 1 mL of RIPA buffer (Sigma) and total protein was extracted according to manufacturers recommendations. Extracted total protein from lung homogenates was quantified using BCA Protein Assay Kits . IL-13 protein in lung homogenates was analyzed by ELISA (R&D systems).To establish the chimera system, bone marrow was extracted from naïve (i.e. un-infected) BALB/c mice and transferred to age-matched irradiated naïve mice. After reconstitution of hematopoietic cells (8 weeks later), AAD was induced and assessed. Allergic mice (Chimera+Ova groups) had significantly increased AHR (increased transpulmonary resistance  and decrC. muridarum Chimera+Ova) as neonates had no effect on Ova-specific IL-5 or IL-13 release in AAD compared to age-matched, un-infected allergic (Vehicle Chimera+Ova) controls  release from MLN cell cultures, whilst an adult infection had no effect controls . In contcontrols . Reconstcontrols . These rC. muridarum Chimera+Ova) as neonates resulted in a small but significant reduction, whilst bone marrow from mice infected as infants increased the number of MSCs in the airways in AAD, compared to age-matched, un-infected allergic (Vehicle Chimera+Ova) controls  as neonates suppressed transpulmonary resistance during AAD, compared to age-matched, un-infected allergic (Vehicle Chimera+Ova) controls  as neonates suppressed IL-13 production during AAD, compared to age-matched, un-infected allergic (Vehicle Chimera+Ova) controls  from MLN cells, MSC numbers around the airways, transpulmonary resistance and IL-13 levels in the lung were all increased during Ova-induced AAD. Adoptive transfer of bone marrow from mice infected as neonates had no effect on Th2-cytokine release, and suppressed the number of MSCs around the airways, transpulmonary resistance and IL-13 in the lung. Infection of adult mice had minimal effects. Therefore, an infant, but not neonatal, chlamydial lung infection, increases AAD in later-life through mechanisms that involve alterations in either the number or phenotype of hematopoietic cells. This is the first study to show that the age of infection can have differential effects on hematopoietic cells resulting in either enhanced, or suppressed, AAD in later-life.In this investigation we demonstrate that In contrast to the affects on transpulmonary resistance, dynamic compliance was unaffected. The large airways are the major contributors to transpulmonary resistance, whereas dynamic compliance is a measure of the elasticity of the small airways and parenchyma ex vivo 6 day cell culture assay that is subject to day to day variation and results are compared between groups performed at the same time and not between different experiments carried out at different times. It is not as robust as MSC enumeration, lung function and IL-13 protein in the lung, all of which are direct representations of what occurred in vivo.There were no differences in all except one of the features of AAD in chimeras generated from sham-inoculated mice. There were no differences between the allergic controls from the three different age groups (Vehicle+Chimera) in terms of MSC numbers, transpulmonary resistance or IL-13 protein in the lung during AAD. This demonstrates that the bone marrow of the chimeras was reconstituted to the same level. The only difference was in the magnitude of MLN cytokine release into culture supernatants. The MLN culture experiments were performed at different times for the different groups. This assay is an Notably all mice were adults at the time bone marrow was extracted and reconstituted. Mice infected or sham-inoculated as neonates, infants or adults were 9, 12 and 15 weeks old at time of bone marrow extraction and reconstitution, respectively. Bone marrow was reconstituted into age-matched controls . Each infected group was compared to its relevant age-matched sham-inoculated control, which was run in parallel with each experiment.Chlamydia lung infection drives more severe Ova-induced AAD in later-life For this study we have used an Ova-induced model of AAD, which involves systemic sensitization and airway challenge. Asthma does have a systemic component although it manifests in the respiratory system. We have previously demonstrated that early-life Our results which show that neonatal infection alters hematopoietic cells to suppress, rather than promote the development of AAD in later-life both support and contrast with what we observed in intact (i.e. wild type) mice in our previous studies Neonatal infection did not alter the release of IL-5 or IL-13 from Ova-stimulated MLN cells but reduced MSC numbers and AHR, which was associated with attenuated IL-13 production in the lung. It is likely that reductions in IL-13 production in the lung contribute to the reductions in MSC numbers and AHR. Potential explanations for these differential effects in MLNs and lungs are that infection may have differential effects on different compartments so that T-cells in the MLNs can still release sufficient amounts of IL-5 and IL-13, but they may not be able to migrate to the lung to induce MSC hyperplasia and AHR. Another explanation could be that systemic DC and/or T-cell function could be altered. Neonatal infection-induced changes in lung structure in intact wild-type mice is also likely to contribute to infection-induced severe AAD In contrast to what occurred in neonates, infection of infants altered hematopoietic cells to increase the severity of AAD in later-life by increasing Th2 (IL-5 and IL-13) cytokine release from MLN cells, MSC numbers, transpulmonary resistance and IL-13 production in the lung. This is consistent with what occurs in intact mice, where infection of infants increased the release of the pro-asthmatic Th2 cytokines IL-5 and IL-13, which was associated with increases in MSC numbers and AHR C. muridarum infection in infants may induce TLR, cytokine and immune cell responses that may promote permanent alterations in the phenotype or activity of hematopoietic cells that drive the development of severe AAD. These possibilities are the subject of ongoing investigations.There is mounting evidence that supports a role for infections in altering hematopoietic cells. Hematopoietic progenitors express TLRs that enable these cells to directly sense bacterial components C. muridarum lung infection in infancy promotes permanent alterations in hematopoietic cells that drive the development of more severe AAD in later-life. Further studies into the effect of respiratory chlamydial infection on hematopoietic cell differentiation and function, as well as elucidating the role of TLR and cytokine responses, will provide a mechanistic insight into how respiratory chlamydial infections in early-life may increase the severity of AAD in later-life. Such studies may identify new therapeutic targets for the prevention of infection-associated severe asthma.In summary,


Repeat radiation is a rarely used treatment strategy that must be performed with caution. The efficacy and toxicity of a second curative radiotherapy series was investigated in cases of recurrent breast cancer.Forty-two patients treated from 1993 to 2003 with resection (n = 30) and postoperative re-irradiation or definitive re-irradiation (n = 12) for recurrent breast cancer were enrolled in the study. Concurrent hyperthermia was performed in 29 patients. The median age was 57 years. The median pre-radiation exposure was 54Gy. Re-irradiation was conventionally fractionated to a median total dose of 60Gy.After a median follow-up of 41 months (range 3-92 months) higher graded late toxicity > G3 according to CTC 3.0 and LENT-SOMA was not observed. The estimated 5-year local control rate reached 62%. The estimated 5-year overall survival rate was 59%. Significantly inferior survival was associated with recurrence within two years .01) and presence of macroscopic tumour load .Repeat radiotherapy for recurrent breast cancer with total radiation doses of 60 Gy and the addition of hyperthermia in the majority of patients was feasible, with acceptable late morbidity and improved prognosis, particularly in patients with previous resection of recurrent tumours. Treatment of pre-radiated regions remains a therapeutic challenge for breast cancer, particularly as the curative potential of local  and systemic  treatment of loco-regional recurrences has yet to be elucidated ,2. AfterTreating chest wall recurrences after mastectomy with re-surgery alone provides limited local control for one-third of patients . The rouNo definitive trial investigating the options of re-treatment i.e. re-irradiation +/- re-surgery has been carried out, and is never likely to be done in view of the aforementioned concerns. Unequal pre-radiation exposure (40-70 Gy) would lead to considerable different risks of late effects within the study population and distort results. Therefore, this retrospective study was performed to evaluate the long-term results and toxicity of local treatment by re-irradiation +/- preceding macroscopically complete resection with curative intent. Furthermore, this analysis focused on tumour- and treatment-related parameters to optimize management of breast cancer recurrences in the future.Between 1993 and 2003, 42 women with locoregional recurrent breast cancer were treated with a macroscopically complete resection plus subsequent re-irradiation of the chest wall (n = 30) or definitive radiotherapy (n = 12) at the University of Tübingen.For inclusion in this retrospective analysis, patients had to fulfill the following criteria: All patients had been initially treated for invasive breast cancer using mastectomy or breast conserving surgery, both combined with adjuvant radiotherapy of the whole breast or the chest wall including regional lymphatics, or a radiation boost if indicated. Patients with preceding or simultaneous metastases, new primary tumours unrelated to their breast cancer or receiving palliative treatment were excluded. The patients fulfilling these criteria were identified by the departmental database. After discussing the intended analysis, the institutional review board  had no objections (Nr. 116/2011A).A brief summary of the clinical characteristics at first diagnosis of breast cancer is provided in Table In-breast recurrences were treated by mastectomy. Chest wall recurrences were removed by local excision- if possible. Re-irradiation was not routinely performed in cases of resected recurrence as an "adjuvant" procedure. Preconditions of individual re-irradiation were close (≤0.5 cm) or positive margins, perinodal involvement, multiple recurrences or other high-risk features explaining a number of recurrences totaling five until re-irradiation was administered.Details regarding second treatments and the course of disease at the time of recurrence are displayed in Table In cases of close or positive margins and definitive treatment, superficial radiofrequency hyperthermia was offered as an additional modality, leading to concurrent application in 29 patients (69%). Almost all patients with microscopically positive margins received hyperthermia , while after R0-resection  or in cases of inoperable recurrences , approximately three-fifths of patients received hyperthermia. Superficial hyperthermia was performed with the SA-115 applicator twice a week using a BSD2000 hyperthermia system operating at a frequency between 210-219 MHz. Median hyperthermia time was 75 min, i.e. 5-15 min heating period and at least 60 min treatment time (steady state of temperature). The target temperature was a minimum of 40°C and measured at the skin.Hyperthermia was performed in accordance with the ESHO-Guidelines. A detailed description of hyperthermia with SA-115 has been published . FurtherAfter re-treatment, senological examinations were performed every three to six months during the first two years and every six to twelve months thereafter. These investigations included clinical examination and imaging if required. Patients were monitored at least annually in terms of toxicity by a radiation oncologist. The Patient's records and questionnaires were reviewed with respect to radiation-induced side effects. Acute radiation dermatitis was investigated during and after re-irradiation and documented according to CTC 3.0. Late toxicity was defined as side effects occurring three months after treatment. Radiation dermatitis, induration/fibrosis, teleangiectasia, pericarditis, pericardial effusion and pneumonitis were investigated according to CTC 3.0. In addition, side effects related to skin (fibrosis/teleangiectasia) and lung (fibrosis) were documented according to LENT-SOMA.Local failure was defined as any recurrence of tumour in the ipsilateral chest wall or in mastectomy scars. Regional failure was defined as any recurrence of tumour in the ipsilateral regional nodes. Recurrence at any other site was considered as distant failure. Time to any failure until re-irradiation was defined as the time from definitive surgery to the time of diagnosis of the first failure. Time to any failure after the second radiation course was defined as the time from start of re-irradiation, in order to compare patients with and without excision.Local control, distant-disease-free survival, disease-free survival and overall survival were the main endpoints, and these were calculated from time of re-irradiation using the Kaplan-Meier method . FurtherThe median follow-up from second radiation treatment was 41 months range 3-92 months). One patient was lost to follow-up. For the 28 surviving women, the median follow-up reached 48 months. With respect to total dose, patients completed radiation treatment as planned. For one patient, a treatment break of two weeks was required owing to acute radiation dermatitis. Concurrent hyperthermia was offered to all patients but some had contraindications  and others refused hyperthermia, leading to an omission rate of 31%. Hyperthermia was completed as planned in 72% of patients (n = 21/29). In two patients, the number of planned hyperthermia treatments was not given. In six patients, the duration of one hyperthermia treatment in each case was shorter than 60 min. Repeat radiotherapy was performed at first recurrence in median but 19 patients experienced at least two recurrences until re-irradiation was administered. Therefore, the median time to second radiotherapy (53 months) was 20 months longer than time to first recurrence . However, local control decreased significantly from 91% to 31% (p = 0.02) if at least two recurrences were experienced before re-irradiation was administered. A time period of less than two years to the first recurrence  did not significantly lower local control. However, long-term local control at last follow-up improved to 70% (n = 35/42) owing to curative resections of recurrences after re-irradiation in three patients. In one of the three patients additional brachytherapy was performed to a total dose of 30 Gy. A sub-analysis of concurrent hyperthermia for R1-resected patients revealed a prolonged local control  but this was not significant. No other investigated factors significantly affected local control (data not shown).The estimated five year overall survival of patients was 59%. Estimated overall survival was significantly shortened in patients who had not undergone surgery of recurrence , Figure Furthermore, the relevance of tumour-related parameters was investigated. Significantly poorer overall survival was detected in women (n = 15) where a time period of less than two years had passed before the first recurrence , Figure Regional (n = 3) and distant (n = 12) metastases manifested in 15 patients.. The mean distant disease-free survival time exceeded more than five years : 46-71 months). The mean disease-free survival was reached before 5 years .No treatment related deaths occurred. Acute and late skin toxicity of radiation schedules are documented in Table Local breast cancer recurrences are a therapeutic challenge with respect to available treatment options, morbidity and toxicity. Depending on the risk features, local recurrence might further herald distant metastasis . The optThis series clearly demonstrates that the best long-term local control was achieved in patients treated with a combined schedule i.e. surgery and re-irradiation predominately performed with hyperthermia and combined with anti-hormonal treatment. Reducing the risk of local relapse using endocrine therapy is in line with findings concerning primary treatment of breast cancer ,20. EighIn 1999, the working group reported a three year local control rate of 80% for complete resections of breast cancer recurrences (most without pre-radiation) after a radiation schedule of 4 × 2.5 Gy/week to a total dose of 50 Gy. This resulted in local control of only 37% for R1/2-resections despite an additional boost to a total radiation dose of 60 Gy . To inteProbably as a result of manifold and overlapping parameters and small sample size of subgroups, local control and survival parameters were improved, but not significantly, by concurrent hyperthermia in this series. The role of hyperthermia after R0-resection remains unclear in this retrospective series. However, it can be assumed that additional hyperthermia compensated for positive margin (R1).In the unfavourable situation of no surgical options, patients have the greatest incremental gain in complete response by additional hyperthermia according to a recently published randomized trial (23.5% vs. 68.2%) . A pooleA recently published multi-institutional review of re-irradiation achieved a 53% 1-year-disease-free survival for unresected patients. Hyperthermia and chemotherapy were applied simultaneously to more than half the women with a median total dose reaching 48 Gy [The only significant tumour-related parameter for overall survival was time below two years to first recurrence. This finding has been substantiated by others ,29. A trIn this series, one of the highest reported median re-irradiation- and cumulative radiation dose was administered. Owing to a conventionally fractionated regimen acute and early late toxicity was moderate as indicated by late effects not exceeding grade 3. This observation is consistent with other retrospective series that reach lower cumulative doses ,32. HoweStandard treatment for breast cancer recurrences is surgical resection, if possible. In general, owing to the detrimental effect of recurrences, early re-irradiation is recommended at first recurrence and a fortiori in case of recurrence within two years, as both factors were associated with dramatically reduced local control and overall survival.Repeat radiotherapy for breast cancer recurrence, with total radiation doses of 60 Gy and the addition of hyperthermia in the majority of patients is feasible. This treatment has acceptable late morbidity and results in improved prognosis particularly in patients who have undergone previous resection of the recurrence.The authors declare that they have no competing interests.ACM: Substantial contributions to acquisition, analysis and interpretation of data, drafting the article and final approval. FE: Substantial contributions to interpretation of data, drafting and revising the article and final approval. VH: Substantial contributions to design, revising the article critically and final approval. MB: Substantial contributions to conception, revising the article critically and final approval. SB: Substantial contributions to interpretation, revising the article critically and final approval. TH: Substantial contributions to conception, analysis and interpretation of data, drafting the article and final approval.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/197/prepub


A low body mass index (BMI) at antiretroviral therapy (ART) initiation is a strong predictor of mortality among HIV-infected adults in resource-constrained settings. The relationship between nutrition and inflammation-related serum biomarkers and early treatment outcomes  in this population is not well described.2 or CD4+ lymphocyte counts of less than 50 cells/mm3, or both, was followed prospectively during the first 12 weeks of ART. Baseline and serial post-treatment phosphate, albumin, ferritin and highly sensitive C-reactive protein (hsCRP) serum levels were measured. The primary outcome was mortality.An observational cohort of 142 HIV-infected adults in Lusaka, Zambia, with BMI under 16 kg/mLower baseline phosphate and albumin serum levels, and higher ferritin and hsCRP, were significantly associated with mortality prior to 12 weeks , independent of known risk factors for early ART-associated mortality in sub-Saharan Africa. The time-dependent interval change in albumin was associated with mortality after adjusting for the baseline value , but changes in the other biomarkers were not.The predictive value of serum biomarkers for early mortality in a cohort of adults with malnutrition and advanced HIV in a resource-constrained setting was primarily driven by pre-treatment values, rather than post-ART changes. Interventions to promote earlier HIV diagnosis and treatment, address nutritional deficiencies, and identify the etiologies of increased systemic inflammation may improve ART outcomes in this vulnerable population. A reducsettings -12.2) and/or immunosuppressed (CD4+ lymphocyte count below 50 cells/mm3) adults initiating treatment in Lusaka, Zambia [We previously demonstrated that serum phosphate levels at ART initiation are independently and negatively associated with early mortality in an observational cohort of severely malnourished  changes in inflammatory biomarkers or albumin, and subsequent health outcomes in persons living with HIV ,17, but 2 or a CD4+ lymphocyte count below 50 cells/mm3 initiating ART at a public sector clinic in Lusaka, Zambia, in an observational, prospective cohort study to assess metabolic predictors of all-cause mortality in the first 12 weeks of treatment. The study setting, eligibility criteria, design and procedures have been previously described [+ lymphocyte count below 200 cells/mm3, or WHO stage 3 disease and a CD4+ lymphocyte count below 350 cells/mm3); were intending to start therapy the same day; met the BMI and/or CD4+ threshold criteria; and agreed to adhere to the study visit schedule and laboratory testing requirements.We enrolled 142 HIV-infected adults with a BMI of less than 16 kg/mescribed . BrieflyAt the enrolment visit and the subsequent study visits at one, two, four, eight and 12 weeks post-ART initiation, participants were evaluated by a research nurse and a clinical officer and/or a supervising physician. The first-line ART regimen was selected from the national programme formulary: two nucleoside reverse transcriptase inhibitors in combination with one non-nucleoside reverse transcriptase inhibitor.At the initial visit, a detailed health history, review of systems, physical examination and laboratory testing  were performed. Phosphate and albumin were measured at each subsequent study visit, while ferritin measurements were repeated at one, two, four and 12 weeks, and highly sensitive C-reactive protein (hsCRP) measurements were repeated at one, four and 12 weeks post-ART initiation. Thirteen participants received phosphate supplementation according to predetermined algorithm based on serum level .Participant deaths were reported to the study staff by the health clinic. If a participant missed a study visit and could not be contacted by phone, community outreach teams attempted to locate the individual or a close relative to determine the vital status . If the + lymphocyte counts were performed using a Beckman Coulter Epics XL-MCL flow cytometer , and hemogram using a Horiba ABX Pentra 80 .Routine and study-specific chemistry assays were measured using a Roche COBAS Integra 400+  or a Pointe 180 Chemistry Analyzer . CD4+ lymphocyte count and BMI to extract the first principal factor to represent these variables at baseline. All Cox models were adjusted for sex and this principal component.Cox proportional hazards models were used to assess the relationship between the baseline nutrition or inflammation serum biomarker values and time to death. The biomarkers were modelled as continuous variables and expanded using restricted cubic splines to avoid linearity assumptions. To reduce the number of variables in the Cox models, we used a principal component analysis with age, hemoglobin, CD4If the vital status at 90 days was unknown , the participant was censored at the last study visit date. A second multivariable regression used similar techniques to model the relationship between the baseline biomarkers and a composite endpoint of time to death or loss to follow up. We accounted for missing baseline status values  and serum biomarkers  using multiple imputation techniques . The proTo visualize longitudinal data, we plotted the serum indicators of interest for the alive, deceased and deceased/lost cohorts against the number of days on ART; each participant required a minimum of two recorded values for inclusion . For each variable, we sketched a locally weighted scatterplot smoothing (LOWESS) curve by fitting a polynomial surface using local (weighted) least squares regression .http://www.r-project.org) was used for data analyses.Cox models with baseline and time-dependent covariates were used to assess the relationship between serial biomarker values and time to death, or the composite endpoint of time to death or loss to follow up. Each biomarker was modelled separately, and the model was adjusted for the biomarker baseline serum value, in addition to sex and the principal component . If the participant had no recorded serum level within a given time interval , he or she was dropped from the risk set for that interval. R-software 2.11.0 , and the Institutional Review Boards at the University of Alabama at Birmingham  and Vanderbilt University .2 and 110 (77%) had CD4+ lymphocyte counts below 50 cells/mm3 . Twenty-five participants died over the 12-week follow-up period ; 10 (40%) participants died within four weeks of starting ART, although none died in the first week. The median time to death was 34 days . Thirty-three participants (23%) were lost to follow up; the median follow-up time for those lost was 58 days . Table We enrolled 142 participants between 6 November 2006 and 12 November 2007; 59 (42%) participants had BMI below 16.0 kg/mTable Conversely, higher baseline serum ferritin and hsCRP levels were associated with increased mortality; compared with a ferritin value of 25 μg/L, a baseline serum level of 250 μg/L was associated with an approximate 67% increased hazard of mortality at 12 weeks , and a value of 1000 μg/L was associated with a nearly 10-fold increased hazard . A baseline hsCRP level of 15 mg/L was associated with a nearly two-fold increased hazard of death compared with a value of 5 mg/L .All adjusted hazard ratios were significantly different from zero (p < 0.05). The calculated hazard of reaching the combined endpoint of mortality or loss to follow up for each gradation of the serum indicators was less pronounced compared with the hazard of mortality alone, and the association was not statistically significant for phosphate or hsCRP.Figure Table For interval increases in phosphate, ferritin and hsCRP, the adjusted hazard ratios demonstrated a trend in the same direction as the hazard ratios for interval changes in the baseline biomarker values Table , but theWe previously reported that baseline serum phosphate predicted early mortality in our cohort , so we iWe also repeated the time-dependent Cox analyses by "carrying forward" the last recorded laboratory value for each serum biomarker until the next recorded value or until death, loss to follow up or study termination occurred . An interval increase in albumin remained a significant predictor of mortality and the composite endpoint  while interval changes in phosphate, ferritin and hsCRP remained non-significant.We found that pre-treatment nutrition and inflammation serum biomarker levels were associated with mortality prior to 12 weeks among adults with advanced malnutrition and/or immunosuppression initiating ART in a resource-constrained setting. A post-treatment interval change in albumin was also a significant predictor of early mortality, while changes in phosphate, ferritin and hsCRP were not. Our study is the first to describe the relationship of these biomarkers to survival in the immediate post-ART period among patients in the advanced stages of HIV disease, but our findings support similar prior studies investigating longer term health outcomes ,17,22.These findings do not imply a causal relationship between elevated inflammation biomarkers or low phosphate or albumin and health outcomes, as individuals with untreated HIV and advanced malnutrition likely have multiple complex metabolic and physiologic derangements. Systemic inflammation in the setting of HIV infection may be a response to occult opportunistic infections, HIV-1 replication, immune reconstitution inflammatory syndrome, or other conditions. Elevated ferritin, in particular, may principally represent a reaction to infectious agents. The pathophysiologic processes contributing to mortality in the immediate post-ART period are a critical research uncertainty and likely differ from those present after several months or years on treatment.Prior studies have reported greater baseline immune activation in cohorts of HIV-infected and non-infected African adults compared with similar groups in the US and Europe -25. ThisWe previously reported that the absolute value of one-week serum phosphate and the change from the pre-treatment level were not significantly associated with mortality in this cohort, as might be observed in the refeeding syndrome . This coRelative long-term  declines in serum albumin are associated with increased mortality among HIV-infected women (both with and without ART) , but abs+ lymphocyte count, BMI and hemoglobin, but residual confounding and/or other unmeasured variables may have affected the associations between serum biomarkers and mortality. Our study investigated relatively few biomarkers of inflammation and nutrition; future studies of early mortality should include a wider range of variables .The time-dependent analyses of interval changes in phosphate, ferritin and hsCRP demonstrated non-significant hazard ratios that uniformly trended in the same direction as the ratios for the baseline values, suggesting that our study cohort was potentially not large enough to detect a true association. Additionally, our analyses adjusted for potential confounding variables, including sex, age, CD4Our cohort also had a high rate of participant attrition, but the observed loss rates are similar to reports from other programmatic cohorts in sub-Saharan Africa . It is lIn this observational cohort study, pre-treatment phosphate, albumin, ferritin and hsCRP serum levels were associated with mortality in the first 12 weeks on ART, but only the interval change in post-treatment albumin was associated with this outcome, while changes in the remaining biomarkers were not. The presence of increased baseline inflammation biomarkers among those deceased at 12 weeks may indicate the presence of undiagnosed secondary infections, such as tuberculosis, fungi and parasites.Measurement of pre-treatment phosphate, albumin, ferritin and hsCRP serum levels may have a role in the risk stratification of low BMI adults starting ART, and in the future, could prompt additional diagnostic procedures, trials of anti-inflammatory therapies, nutritional supplementation, or other targeted intervention strategies. The role of post-treatment monitoring of phosphate and inflammation biomarkers should be explored in larger studies.Low BMI individuals represent a considerable proportion of adults requiring HIV care in resource-constrained settings, and further study of the metabolic and physiologic derangements, co-morbid infections, and potential therapeutic interventions to improve outcomes in this vulnerable population are necessary.The authors declare that they have no competing interests.DH, CN, EK, IZ and LM were responsible for study design and data collection. MB, BS and JK performed the statistical analyses. JC and EK performed the laboratory analyses. JK, MB, EK, CW, JC and AM drafted the manuscript, which all authors reviewed, edited and approved.


Epidermal growth factor receptor (EGFR) inhibitors are widely used in the treatment of cancer. EGFR-targeted treatment is known to be associated with a high incidence of dermatological adverse reactions, including papulopustular rash, which can be dose-limiting and may affect compliance to treatment. Currently, the pathways involved in EGFR inhibitor-induced rash are poorly understood and few treatment options for this adverse event are available. Here, we developed a model for induction of papulopustular rash in healthy human volunteers by subcutaneous injection of the anti-EGFR monoclonal antibody zalutumumab. The injection sites and surrounding skin were evaluated by a dermatologist for the presence or absence of papulopustular rash and skin biopsies were taken to confirm the macroscopical findings by immunohistochemistry. Locally injected zalutumumab induced a papulopustular rash, characterized by acute follicular neutrophil-rich hair follicle inflammation, and thus mimicked adverse events induced by systemic administration of EGFR inhibitors. In this model, we tested the hypothesis that neutrophils, attracted by IL-8, play a central role in the observed rash. Indeed, concomitant local repeat dose treatment with HuMab-10F8, a neutralizing human antibody against IL-8, reduced the rash. Inhibition of IL-8 can therefore ameliorate dermatological adverse events induced by treatment with EGFR inhibitors. Cancer therapy is increasingly shifting towards targeting specific pathogenic pathways. Epidermal growth factor receptor  controls proliferation and maturation of epithelial cells in skin. In many solid tumors of epithelial origin, EGFR is up-regulated, making it an attractive target for treatment The rash induced by EGFR inhibitors typically appears within one to three weeks of treatment and is characterized by inflammatory follicular papules and pustules. The rash is most commonly affecting the face; but is also seen at the upper chest and back and infrequently at other body sites The rash may be related to follicular occlusion due to a lack of epithelial differentiation and epithelial inflammation resulting from release of cytokines as direct results from EGFR inhibition. Because the papulopustular rash is characterized by follicular inflammation with an accumulation of neutrophils Here we show, in this proof-of-principle study, that inhibition of IL-8 can ameliorate the dermatological adverse events induced with an EGFR-inhibiting mAb. Further studies addressing the potential of IL-8 inhibition for the prevention of serious dermatological adverse events induced both by small molecule as well as biologic EGFR inhibitors are warranted.An open-label, single-center non-randomized study was performed in healthy volunteers with a single dose escalation set-up. The clinical study was performed at the Department of Dermato-allergology, University Hospital of Copenhagen Gentofte in accordance with the declaration of Helsinki. The study was approved by the local ethics committee (H-KA-20060104) and The Danish Medicines Agency (2006-003253-24). All subjects gave written informed consent prior to enrolment.A total of nine healthy male volunteers were included in the study. All subjects were Caucasian men and the median age of the group was 24 years (range 22–32 years).The first part of the study was conducted to evaluate whether local subcutaneous (s.c.) injection of zalutumumab could induce a papulopustular rash, similar to that reported in patients treated systemically with EGFR inhibitors. A maximum of four subjects were to be enrolled and attended once weekly for injection of escalating doses of zalutumumab on the upper back. Since there was no experience with s.c. injection of zalutumumab and the local concentration to induce rash was not known, the study was started with a dose-escalation of s.c. zalutumumab see . 1 μg  HuMab-10F8. As a negative control, isotonic saline, in the same volume as the combined zalutumumab/HuMab-10F8 injection (0.45 mL), was injected at a different site at each visit. After the fourth injection of the zalutumumab/HuMab-10F8 combination at one dose level, 1.25 mg HuMab-10F8 was injected alone once weekly for three weeks at the combination injection site see .In the third and last part of the study, zalutumumab was given in a dose-escalation procedure starting at 100 μg, or 1 mg if the volunteer had received 100 μg earlier in the study. HuMab-10F8 was administered in a repeat dose regimen. Four subjects were enrolled. The procedure was as described for the second part of the study; however, an additional 1.25 mg dose of HuMab-10F8 was administered at four consecutive days after each combined zalutumumab/HuMab-10F8 injection.The therapeutic antibodies used in this study were zalutumumab ; a human IgG1, κ mAb specific for EGFR; generated as described in Bleeker et al., by immunizing HuMAb mice with A431 cells and purified EGFR In each study part, injection sites were evaluated once a week and subjects were instructed to contact the investigator as soon as pustules developed. 4-mm punch skin biopsies were taken from a pustule when a rash was considered to be induced by zalutumumab treatment. At the same time, control biopsies were taken from the isotonic saline injection site and, in part two and three of the study, the positive control site .The investigator assessed the degree of the rash at the marked injection sites using the following scale from 0 to 3: 0: no rash; +1: mild rash; +2: moderate rash; +3: severe rash. The number of pustules at the injection site was counted and photographic documentation obtained.Paraffin-embedded skin biopsies were cut at 4 μm thickness, stained with HE and scored as normal or acutely inflamed. Multiple skin sections of each biopsy were examined, to secure that areas with inflammation deeper in the biopsies would not be missed. HE-stained skin biopsies were scored normal when only few diffuse macrophages or lymphocytes were detected in the vicinity of hair follicles (sebaceous glands) and scored acutely inflamed when, in addition to macrophages and lymphocytes, influx of neutrophils was detected . To confirm the conclusions of the HE staining and identify the inflammatory cells, some skin biopsies were immunostained for EGFR , or with cell type-specific markers identifying neutrophils  and macrophages . The primary mouse antibodies were detected with a peroxidase-labeled anti-mouse IgG dextran polymer  and visualized with AEC (red staining).To set up a human model to study EGFR inhibitor-induced rash, healthy subjects were injected s.c. with the human anti-EGFR antibody zalutumumab. Local injections were used to set up a safe model in human volunteers with limited exposure to the therapeutic antibody.In part I of the study , Table 1In part II of the study, it was investigated whether neutralization of IL-8 by a neutralizing antibody, HuMab-10F8, could inhibit the zalutumumab-induced rash. Zalutumumab and HuMab-10F8 were administered concomitantly, once weekly . Seven sOverall, five subjects experienced at least one adverse event . Headache (two subjects), influenza (one subject), heamatochezia (one subject) and neutrophil count decrease (one subject) were judged as not related to zalutumumab treatment; rash distant from the injection site (one subject), fatigue (one subject), pharyngitis (one subject), acne (one subject) and pruritus (one subject) were judged as related to zalutumumab treatment. No serious adverse events were reported.Prevention and management of EGFR inhibitor-induced dermatological toxicities is critical for patients on such treatments. Histologically, the papulopustular skin rash of patients systemically treated with EGFR inhibitors for cancer often presents with neutrophil-rich suppurative folliculitis Papulopustular rash is reported in up to 80% of patients treated with EGFR inhibitors, both small molecules and mAb in vivoEGFR is expressed on epidermal keratinocytes, hair follicle epithelium and sebaceous glands in vitroin vitro to a whole organ in vivo, in this case the skin, which contains many different cell types that interact with each other, and may respond distinctly. Our exploratory clinical trial allowed us to test our hypothesis in humans in vivo.EGFR blockade has been demonstrated to affect chemokine expression in keratinocytes Previously, we showed that local injection of HuMab-10F8, a therapeutic human antibody against IL-8, induced a marked reduction of disease activity in PPP patients In addition to a role in inflammation, IL-8 was also demonstrated to play a role in the survival of tumor cells. Blocking IL-8 has been shown to prevent angiogenesis and reduce tumorigenesis of human non-small cell lung cancer in SCID mice In summary, we developed an innovative human model to study dermatological adverse events induced by EGFR inhibitors. Using this model we showed that neutralization of IL-8 by specific mAb is capable of ameliorating these adverse events. Our study provides a proof-of-principle for a role of IL-8-induced neutrophil migration in the biological mechanisms involved in EGFR inhibitor-induced rash.


Acupuncture and electro-acupuncture (EA) are now widely used to treat disorders like pain. We and others have shown previously that current frequency, intensity and treatment duration all significantly influence the anti-nociceptive effects of EA. There is evidence that stimulating sites also affect the antinociception, with EA applied ipsilaterally to the pain site being more effective under some pain states but contralateral EA under others. It was recently reported that local adenosine A1 receptors were responsible for ipsilateral acupuncture, but what mechanisms specifically mediate the anti-nociceptive effects of contralateral acupuncture or EA remains unclear. In the present study, we applied 100 Hz EA on the ipsi- or contra-lateral side of rats with inflammatory pain induced by intra-plantar injection of formalin, and reported distinct anti-nociceptive effects and mechanisms between them. Both ipsi- and contra-lateral EA reduced the paw lifting time in the second phase of the formalin test and attenuated formalin-induced conditioned place aversion. Contralateral EA had an additional effect of reducing paw licking time, suggesting a supraspinal mechanism. Lesions of rostral anterior cingulate cortex (ACC) completely abolished the anti-nociceptive effects of contra- but not ipsi-lateral EA. These findings were not lateralized effects, since injection of formalin into the left or right hind paws produced similar results. Overall, these results demonstrated distinct anti-nociceptive effects and mechanisms between different stimulating sides and implied the necessity of finding the best stimulating protocols for different pain states. Acupuncture, applied by inserting long needles into specific points in the body (acupoints), has been used in China for over two thousand years to treat a variety of diseases including pain. Electro-acupuncture (EA), manipulated by passing electric currents through acupuncture needles, is also widely used and allows a more objective control over stimulating parameters. EA activates/deactivates a variety of brain regions and promotes the release of endogenous opioid peptides, which are responsible for mediating its analgesic effects ,2. Work To answer this question, we first established the classic acute inflammatory pain model of rats by intra-plantar injection of formalin, and applied 100 Hz EA on the ipsi- or contra-lateral side of the injected hind paw Figure .Pervious studies have established dissociation between the sensory-discriminative and the emotional-affective dimensions of pain ,11. LesiThe paw licking behaviour in the formalin model parallels neural activities in the rat forebrain , which lWe tested whether lesions of rostral ACC, a forebrain region crucial for pain affection ,11, woulBilateral rostral ACC lesions Figure  induced All the above experiments were performed with formalin injection in left hind paws. To exclude the possibility that these findings were lateralized to one side, we repeated the lesion experiment in a new cohort of rats with formalin injection in their right hind paws and obtained similar results Figure  and 3E. How ACC mediates the anti-nociceptive effects of contralateral EA requires further investigation but several potential mechanisms are proposed here. Nociceptive neurons in ACC have large bilateral receptive fields that often include the whole body ,18, whicAdditionally, the mechanisms of the formalin test's two phases are different: the first phase results from the activation of peripheral nociceptors whereas the second phase reflects the development of inflammation and central sensitization. In our study, EA only attenuated nociceptive behaviours in the second phase of the test, suggesting a potential mechanism of desensitization, which has been reported in the spinal dorsal horn . A similOverall, the present study indicated distinct anti-nociceptive effects and mechanisms between ipsi- and contra-lateral EA. ACC is crucial for the latter but not the former type of EA antinociception. These data support the traditional acupuncture theory that different acupoints should be selected for different pain states . HoweverAdult Sprague-Dawley rats weighing 160-220 g at the beginning of the experiment were provided by the Department of Experimental Animal Sciences, Peking University Health Science Center. All animal experimental procedures were conducted in accordance with the guidelines of the International Association for the Study of Pain and were approved by the Animal Care and Use Committee of the University. The behavioural experimenters were kept blind from the groupings of the rats.All subjects were divided into three groups: the formalin group, which received formalin injection in their left hind paws without EA treatment, and the ipsi- and contra-lateral EA groups, which received EA application on their left and right leg before formalin injection, respectively Figure . For EA Immediately after EA application or restriction, each rat was administered a 0.1 ml injection of 8% formalin solution into the plantar surface of the left hind paw. After injection, the rat was placed into a 30 × 30 × 30 cm Plexiglas chamber. Behavioural testing immediately began and lasted for 60 min. The amount of time the animal spent with the injected paw lifted or licked was recorded, respectively  for the Animals were trained in a black-walled shuttle box  consisted of three interconnected compartments  with distinctive visual and tactile cues. A consecutive 4-day training procedure was used. On day 1 (pre-conditioning day), each rat was allowed free access to all three compartments for 15 min while the time spent in each compartment was recorded to assess its innate place preference. On day 2, rats were randomly restrained in the left or right compartments for 45 min. On day 3, rats received no treatment (sham group), or an injection of formalin  into the left hind paw  and were then counterbalanced with 45-min restraint in the opposite compartment . Rats in the ipsi- or contra-lateral EA group received EA treatment in their left or right leg before formalin injection as described above. On day 4 (post-conditioning day), each rat was again allowed free access to all three compartments for 15 min, and the time spent in each compartment was recorded. CPA scores represented the time spent in the treatment-paired compartment on the pre-conditioning day (day 1) minus the time spent in the same conditioning compartment on the post-conditioning day (day 4) . Time inBilateral electrolytic lesions of rostral ACC were performed with a constant current of 1 mA for 20 s to the following stereotaxic coordinates: AP +1.6 mm from bregma, ML ± 0.6 mm, DV 2.0 mm. Animals of sham lesions underwent the same surgical procedure except for passing the current. One week after the surgery, the animals received a left intra-plantar injection of 0.1 ml 10% formalin solution. Six groups were included: sham lesion, sham lesion + ipsilateral EA, sham lesion + contralateral EA, ACC lesion, ACC lesion + ipsilateral EA, and ACC lesion + contralateral EA. Formalin was injected in left or right hind paw in two independent cohorts of animals. EA was applied as described earlier. Histological confirmation of ACC lesions was performed by microscopic inspection of 50 μm coronal brain sections with Nissl staining.ACC: anterior cingulate cortex; EA: electro-acupuncture; F-CPA: formalin-conditioned place aversion.The authors declare that they have no competing interests.MY and ZH contributed equally for this manuscript. MY, LL, GGX and YW raised the hypothesis. MY, HZ, LL, GGX and YW designed the experiment. MY and HZ carried out the experiments and performed the data analysis. MY, HZ and YW drafted the manuscript. All authors read and approved the final manuscript.


Rice stripe virus and described the character of the small interfering RNAs (siRNA) derived from the RSV RNA genome. We now report the identification of novel miRNAs from the abundant sRNAs (with a minimum of 100 sequencing reads) in the sRNA library of RSV-infected rice. 7 putative novel miRNAs (pn-miRNAs) whose precursor sequences have not previously been described were identified and could be detected by Northern blot or RT-PCR, and were recognized as novel miRNAs (n-miRNAs). Further analysis showed that 5 of the 7 n-miRNAs were up-expressed while the other 2 n-miRNAs were down-expressed in RSV-infected rice. In addition, 23 pn-miRNAs that were newly produced from 19 known miRNA precursors were also identified. This is first report of novel rice miRNAs produced from new precursors related to RSV infection.MicroRNAs (miRNAs) play essential regulatory roles in the development of eukaryotes. Methods based on deep-sequencing have provided a powerful high-throughput strategy for identifying novel miRNAs and have previously been used to identify over 100 novel miRNAs from rice. Most of these reports are related to studies of rice development, tissue differentiation, or abiotic stress, but novel rice miRNAs related to viral infection have rarely been identified. In previous work, we constructed and pyrosequenced the small RNA (sRNA) libraries of rice infected with  MicroRNAs (miRNAs) are small 19–24 nt RNAs that play essential roles in eukaryotes by targeting complementary mRNAs for degradation or translational repression miRNAs in plants regulate leaf morphogenesis, the development of roots and flowers and other key processes, and are recognized as important regulators of plant development http://www.mirbase.org/cgi-bin/browse.pl) Medicago truncatula, Oryza sativa and Glycine max are the three plants that have the most identified miRNAs  In plants, over 4600 miRNAs have been identified from over 50 species  is the type member of the genus Tenuivirus. It is transmitted by the brown planthopper Laodelphax striatellus in a persistent manner and causes rice stripe disease, which is severe in rice fields in East Asia Until now, most reports of novel rice miRNAs have been related to studies of rice development, tissue differentiation, or abiotic stress, while the novel rice miRNAs related to viral infection have rarely been identified. Identifying the novel miRNAs related to viral infection in rice would be helpful to broaden our understanding of the miRNA response of rice to virus infection in general and is likely to provide a model for other cereal plants in particular. In previous work, we constructed and pyrosequenced sRNA libraries of rice inoculated with RSV and mock-inoculated controls, and described the character of the small interfering RNAs (siRNA) derived from the RSV RNA genomes In our previous work, we constructed sRNA libraries of RSV-infected and non-infected rice by the Illumina Solexa sequencing system http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) and their loci in the genome were recorded. The sRNAs that had more than 26 loci in the rice genome were not considered in the following analysis for two reasons: 1) the highest number of loci of known miRNAs in the rice genome is 26 (osa-miR395); and 2) the higher number of loci more often occurs when the loci are located in the repeat-rich regions of the rice genome; siRNAs but not miRNAs can be produced from the repeat-rich regions that have predicted fold-back structures (miRBase). This largely excludes the contaminative effect of siRNA on miRNA identification.The analysis was done in three steps. In the first step, we aligned the 239 chosen sRNAs with all the mature miRNAs in miRBase to identify the known miRNAs. The recognized miRNA sequences in sRNAs were excluded from the next step. In the second step, the remaining sRNAs were aligned with all the reported miRNA precursor sequences to identify putative miRNAs newly produced from the known precursors. These sRNAs were excluded from the following step. In the third step, the precursor sequences of all remaining sRNAs were predicted using a modification of the reported method http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form). A sequence that has a secondary structure with at least 16 paired nucleotides in its stem region and has free energy less than or equal to −20 kCal/mol was considered to be a putative precursor sequence.For each locus, two sequences respectively extending 200 nt upstream and 20 nt downstream, or 20 nt upstream and 200 nt downstream of the sRNA were extracted for secondary structure prediction. The secondary structures were predicted by the Mfold RNA folding platform  (carrying the RSV-Zhejiang isolate) were transferred onto healthy rice seedlings (Oryza sativa L. japonica. cv. Nipponbare) at the three-leaf stage for virus inoculation. Control seedlings were inoculated with non-viruliferous planthoppers. After 72 h, the planthoppers were removed. Systemic infections were confirmed by RT-PCR specific for RSV Zhejiang isolate RSV-infected rice were prepared as described Total RNA was isolated from the frozen plant materials with Trizol  according to the manufacturer's instructions. 50 µg of DNase-treated total RNA was separated on a 15% polyacrylamide gel, and transferred electrophoretically to Hybond-N+ membranes (Amersham Bioscience) using 20×SSC. Membranes were baked at 80°C for 2 hours. DNA oligonucleotides complementary to the putative miRNA sequences were end-labeled with DIG using the DIG Oligonucleotide 3′-end labeling Kit (Roche). Membranes were pre-hybridized for at least 1 h and hybridized overnight at 42°C using DIG High Prime Labeling and Detection Starter Kit II (Roche). The hybridization signals were visualized by exposure to X-ray film (Kodak).T method. The primers used are listed in We used the published method for RT-PCR of sRNAs with modifications http://plantgrn.noble.org/psRNATarget/) Putative targets for the novel miRNAs were predicted by psRNATarget .The number of reads of each sRNA from RSV-infected rice was divided by the number of reads in the non-infected controls of the same experiment. A two-tailed http://www.mirbase.org/index.shtml), 43 sRNAs that completely matched with the deposited rice miRNAs were identified  recently reported that RSV infection induced different miRNAs to be produced from a single precursor in a phased pattern Among these 23 sRNAs, 12 were produced from the 5′ arm and 11 were produced from the 3′ arm of their respective precursor. Seq114 and 115 were located at the miRNA* region of osa-miR408; Seq116 was located at the miRNA* region of osa-miR167 h; Seq117 was located at the miRNA* region of osa-miR398a; Seq118 was located at the miRNA* region of osa-miR444c. These sRNAs may be the miRNA* or miRNA* isoforms of the corresponding miRNAs.We also searched for these 23 sRNA in the control library and other reported RSV and non-infected rice sRNA libraries, and found that they existed in the libraries with different sequencing reads . StatistThe precursor sequences and corresponding stem-loop structures were predicted for each of the remaining 121 sRNAs that did not match the deposited rice miRNAs or miRNA precursors. 7 sRNAs (Seq119–125) that have precursor sequences capable of forming a stem-loop structure with free energies ranging from −23.5 to −67.9 kcal/mol, and that have no more than 26 hit loci in the rice genome were identified , while tAll 7 sRNAs could also be found with 1–5517 sequencing reads in the non-infected sRNA library reported by us and in 6 rice sRNA libraries reported by Du et al To confirm that the 7 pn-miRNAs were actually produced in plants, we detected them in RSV-infected rice by Northern blots. In three independent experimental repeats, 4 of the 7 pn-miRNAs  could be detected in total RNAs of RSV-infected rice, but the other three pn-miRNAs could not . ConsideThe 7 n-miRNAs analyzed here were identified from the RSV-infected rice sRNA library. To know whether their expression levels were affected by RSV infection, we tried to compare the number of sequencing reads in our reported RSV-infected and non-infected rice sRNA libraries. Considering that plant miRNA target sites are predominantly located in ORFs, we here only focused on finding target sites in coding regions. Seq119, 120, 121 and 123 had one or two targets, while Seq122, 124 and 125 had three or more targets each  the miRNA and antisense miRNA should be derived from the opposite stem arms of the precursor stem-loop structure; 2) the mismatched bases between the miRNA and anti-sense miRNA are restricted to four or fewer; and 3) the frequency of asymmetric bulges in miRNA/miRNA* duplex is restricted to less than one and the size should be less than two bases In the pyrosequenced sRNA libraries, small interfering RNA from double stranded RNAs or decay products of large precursors are usually found at low frequency Deep sequencing has been a powerful method for identifying miRNAs in many plants. In addition, the sequencing reads are thought to reflect the levels of expression of the sequences. Among the n-miRNAs identified here, results from sequencing reads showed that only Seq123 was down-expressed with significant changes in RSV-infected rice. However, real-time PCR results showed that all n-miRNAs had altered expression patterns in the RSV-infected rice: Seq120 and Seq125 were down-expressed in RSV-infected rice, while Seq119, Seq121, Seq122, Seq123 and 124 were up-expressed after RSV infection . Some ofIn the 46 known miRNA families identified, osa-miR167, 168, 528, 172, 444, 166, 535, 164, 396 and 827 were the ten most abundant miRNA families. This is nearly completely consistent with the results of Du et al., in which osa-miR168, 156, 396, 159, 535, 166, 172, 167, 528 and 444 were the ten most abundant miRNA families, indicating stable functions for these miRNAs in RSV-infected rice. Production of new miRNAs from known precursors was also detected here. One or more new miRNAs were identified from each of 19 known miRNAs. Among these 19 known miRNA precursors, these are the first reports of a second mature miRNA from osa-MIR169a, 187a, 1883b, 1884a, 806g, 807b, 808, 809b, 812a, 1439, 812k and 2123c. Most of the newly produced miRNAs have predicted targets, indicating their potential function in rice . MoreoveRSV-induced rice disease is serious in China, Japan and Korea. Virus variation and the functions of the RSV-encoded proteins have been well studied in recent years Figure S1Alignments of each n-miRNA and its cloned sequence. Sequence 1 represents the n-miRNA sequence; Sequence 2 represents the cloned sequence in pGEM-T vector for sequencing, Sequence 3 represents the expected sequence for cloning.(TIF)Click here for additional data file.Table S1sRNAs used for analysis here.(XLS)Click here for additional data file.Table S2Primers used for RT-PCR and real-time PCR(DOC)Click here for additional data file.Table S3Predicted targets for the identified miRNAs newly produced from known miRNA precursors.(DOC)Click here for additional data file.


Calorie restriction  has been associated with substantial alterations in body composition and insulin sensitivity. Recently, several proteins that are secreted by nontraditional endocrine tissues, including skeletal muscle and other tissues, have been discovered to modulate energy metabolism, body composition, and insulin sensitivity. The aim of this study was to characterize the influence of CR by rats on plasma levels of six of these newly recognized metabolic hormones . Body composition of 9-month old male Fischer-344/Brown Norway rats (AL and CR groups) was determined by nuclear magnetic resonance. Blood sampled from the carotid artery of unanesthetized rats was used to measure concentrations of glucose and plasma proteins. As expected, CR versus AL rats had significantly altered body composition  and significantly improved insulin sensitivity (based on the homeostasis model assessment-estimated insulin resistance index). Also consistent with previous reports, CR compared to AL rats had significantly greater plasma levels of adiponectin and corticosterone. However, there were no significant diet-related differences in plasma levels of BDNF, FGF21, IL-1β, myonectin, myostatin, or irisin. In conclusion, these results indicate that alterations in plasma concentration of these six secreted proteins are not essential for the CR-related improvement in insulin sensitivity in rats. Moderate calorie restriction  has well-known effects on body composition, glucose homeostasis, plasma insulin concentration, insulin sensitivity, and other aspects of metabolic health in many species, including rats -3, mice We were especially interested in CR effects on plasma proteins that are secreted into the circulation by skeletal muscle  because insulin signaling in this tissue is highly responsive to CR . CR induThe goal of the current study was to characterize the effects of moderate CR on plasma levels of myonectin, myostatin, irisin, BDNF, FGF21, and IL-1β in 9 month-old rats. These proteins were selected for study because they are newly discovered plasma myokines and/or cytokines that were recently recognized to have metabolic functions, and they have not been previously studied with CR. Accordingly, it seemed possible that they might play a role in the CR phenotype . We also assessed other parameters that have been previously documented to be responsive to CR-induced changes  ,21-24. WProcedures for animal care were approved by the University of Michigan Committee on Use and Care of Animals. Male Fischer-344 x Brown Norway, F1 generation rats were obtained at 3 months of age from Harlan . Rats were housed individually in shoebox cages and maintained on a 12:12 h light–dark cycle (lights out at 17:00 h). After familiarization in the Ann Arbor facility, rats were assigned to either AL or CR groups. At baseline (~ 14 weeks-old) prior to initiating the dietary protocol (CR group received 60–65% of AL intake daily for approximately 6 months as previously described ), the bo-1) x insulin (μU·ml-1)/405] was calculated [ELISA kits  were used to measure plasma levels of insulin (#EZRMI-13K), adiponectin (#EZRADP-26K), BDNF (#CYT306), and FGF21 (#EZRMFGF21-26K). Multiplex bead kits (EMD Millipore) were used to measure C-peptide (#RMHMAG-84K), corticosterone (#RSH69K) and IL-1β (#RCYTOMAG-80K). Reagents and apparatus for SDS-PAGE and immunoblotting were from Bio-Rad Laboratories . Anti-FNDC5 antibody (#ab93373) was from Abcam . Anti-myostatin antibody (#AB3239) was from EMD Millipore. Anti-rabbit IgG-horseradish peroxide conjugate (#7074) was from Cell Signaling Technology . Anti-myonectin antibody was a gift from Dr. G. William Wong at Johns Hopkins University School of Medicine. Myonectin and myostatin in plasma were determined by Western immunoblotting procedure as previously described . Brieflylculated . UnpaireCR rats compared to AL rats had significantly (P ≤ 0.05) lower body mass and absolute values (g) for fat mass, lean mass and free fluids Table . When boThe primary goal of this study was to examine the influence of CR on the plasma concentrations of six proteins that were recently recognized to have metabolic functions  and that had not previously been studied in the context of CR. The results revealed no significant differences for any of these plasma proteins in AL compared to CR rats. It is notable that the CR protocol was accompanied by the expected changes in both body composition (lower percent fat mass and greater percent lean mass) and insulin sensitivity (improvement based on the reduction of the HOMA-IR index). The current results also confirmed earlier reports of CR-induced elevations in the plasma levels of adiponectin . Taken tIt is important to recognize that the current results do not eliminate the possibility that one or more of these plasma proteins may be relevant for some of CR’s metabolic effects. It remains possible that CR may alter their diurnal fluctuations, that CR effects may be localized to protein concentrations in selected cells or tissues, or that CR may modify the sensitivity of target-cells. The current results also do not address the possibility for different outcomes during more brief or severe CR, in animals of other ages, in females, or in other species. Future studies should clarify possible CR effects on the originating tissues and time-courses for the secretion of these proteins and determine if the results for CR found in adult male rats are typical of other populations.The authors declare that they have no competing interests.NS, CMC and GDC participated in the design and coordination of the study. NS and CMC performed the experiments and participated in the data acquisition. NS and GDC participated in the data analysis and drafting of the manuscript. All authors read and approved the final manuscript.


The standard of care in Locally-Advanced Non-Small Cell Lung Cancer (LA-NSCLC) is chemotherapy and radiation; however, Radiation-Induced Lung Injury (RILI), which may be prevented by the anti-inflammatory and anti-oxidant properties of Flaxseed (FS), impedes its maximum benefit.Patients with LA-NSCLC requiring definitive RT were randomized to one FS or control muffin daily from start to 2 weeks after RT. Blood and urine were collected to quantify plasma FS metabolites, Enterodione (ED) and Enterolactone (EL), and urinary oxidative stress biomarkers, 8, 12-iso-iPF2a-VI (isoprostane) and 8-oxo-7,8-dihydro-2′deoxyguanosine (8-oxo-dGuo). Tolerability was defined as consuming ≥ 75% of the intended muffins and no ≥ grade 3 gastrointestinal toxicities.Fourteen patients  were enrolled. The tolerability rates were 42.9 versus 71.4% (p=0.59) for FS and control, respectively. Mean percentages of intended number of muffins consumed were 37% versus 73% (p=0.12). ED and EL increased at onset of FS and decreased with discontinuation, confirming bioavailability. Isoprostane and 8-oxo-dGuo were detectable. There was a trend towards decreased rates of pneumonitis in FS.This is the first study to report FS bioavailability and quantify oxidative stress markers in NSCLC patients. FS in the administered muffin formulation did not meet tolerability criteria. Given the promising mechanism of FS as a radioprotectant, further investigations should focus on the optimal method for administration of FS. Reducing oxidative stress would hypothetically prevent RILI and improve the therapeutic index of radiotherapy in lung cancer. No known effective pharmacologic therapy to prevent this challenging complication of radiotherapy exists.The standard of care for patients with unresectable, locally-advanced Non-Small Cell Lung Cancer (NSCLC) is definitive Radiation Therapy (RT) given with or without chemotherapy. However, outcomes with this modality are quite poor with 5-year survival estimates at 16%  and mani4 and the rate of urinary excretion of 8-oxo-dGuo parallels the rate of oxidative stress. Biochemical studies have revealed that certain populations  have baseline elevations in 8-oxo-dGuo levels that do not trend back to epidemiological baseline levels after an oxidative insult was terminated [in vitro to have direct hydroxyl radical scavenging properties, limit the respiratory burst of White Blood Cells (WBCs) and to inhibit lipid peroxidation [In this study, two urinary biomarkers of systemic oxidative stress, 8, 12-iso-iPF2a-VI isoprostane and 8-oxo-7, 8-dihydro-2′deoxyguanosine (8-oxo-dGuo), were investigated. Isoprostanes, oxidative stress biomarkers, are prostaglandin-like compounds formed by the peroxidation of arachidonic acid by free radicals. Reactive oxygen species (ROS) cause the 8-hydroxylation of guanine (8-oxo-Guo) . This moxidation . Flaxseexidation , cardiovxidation , has helThe primary objective of this study was to ascertain the feasibility of incorporating a validated radioprotector, Flaxseed (FS), in the treatment regimen of patients with advanced NSCLC receiving chemotherapy and thoracic radiation. We specifically focused on whether flaxseed muffins would have a reasonable toxicity and tolerability index for patients receiving chemoradiation. We additionally sought to validate FS bioavailability by quantifying serum markers of metabolism and quantifying urinary markers of systemic oxidative stress before, during, and after RT in the presence and absence of concurrent FS supplementation. An additional component of the study aimed to establish if serum lignan levels and/or urinary markers of systemic oxidative stress demonstrated a dose-response relationship with 20 versus 40 grams of flaxseed.This randomized, double-blinded, placebo-controlled study was conducted in accordance with the IRB. Eligible patients were 18 years or older and had a diagnosis of non-small cell lung cancer (NSCLC) requiring definitive thoracic and mediastinal radiation. The study was amended in February 2009 to require concurrent chemotherapy with RT. Patients with metastatic disease were eligible.Primary outcomes were toxicity and tolerability of dietary flaxseed administration during chemoradiation. Secondary endpoints included urinary biomarkers of systemic oxidative stress and serum levels of flaxseed metabolites enterodione and enterolactone.The following were criteria for ineligibility: prior thoracic RT; Gastrointestinal (GI), liver, or kidney disease that could result in altered metabolism or excretion of the study medication were excluded, including major Gastrointestinal (GI) surgery, ulcerative colitis, regional enteritis, or GI bleeding; administration of an investigational drug, amifostine or Mucomyst (N-acetyl-cysteine) within 14 days; known hypersensitivity to flaxseed (or its metabolites) or wheat, or those who were currently taking flaxseed, flax-containing products, soybeans, soy-containing products, or other herbals or botanicals. Those taking or had taken Vitamin E in excess of 30 IU within the prior 21 days were excluded.We elected to perform this trial as a randomized placebo-controlled trial because the outcome of tolerability is subjective and it would be best to have a control arm, thus minimizing bias. The trial randomized patients to two dose levels. A standard 1:1 randomization method was applied in this trial, the randomization was performed by the General Clinical Research Center, and therefore the investigators and patients were blinded to the randomization. Twelve patients were first randomized to consume one muffin per day  or one Durham wheat muffin (control) per day (6 patients) starting on the day their definitive RT begins and for the duration of their therapy definitive RT until two weeks after completion of RT . If 2 subjects on the first dose level failed to tolerate the diet, then the trial would be suspended and the randomization code broken. If tolerated, then an additional 12 patients were intended to be randomized to a second dose level of two muffins per day (40 g). All patients underwent CT-based treatment planning for RT. Gross Tumor Volume (GTV) was defined as all known sites of disease including pathologically enlarged (≥ 1 cm in short axis) or FDG-avid lymph nodes. Elective nodal radiation was not permitted. Total dose to gross disease was 60–80 Gray (Gy) administered in 2 Gy fractions given daily 5 days per week. The Clinical Target Volume (CTV) was created from the GTV by adding a 1 cm margin. In 11 out of 14 patients, 4D-CT and PET/CT fusion was used for treatment planning. Dose constraints were as follows: volume of lung receiving 20Gy (V20) less than 35%, maximum spinal cord doss less than 45Gy, dose to 50% of cardiac volume (D50) less than 40Gy. The esophagus dose was calculated for each treatment plan but was not a dose-limiting structure.iso-iPF2a-VI [Circulating plasma levels of ED and EL at the four specimen time points were determined by liquid chromatogram phys tandem mass spectrometry (LC/MS/MS) as described earlier ,9 using iPF2a-VI  and 8-oxiPF2a-VI  were meaiPF2a-VI .Patients were asked to log how many muffins were consumed during the study. Toxicity, including pneumonitis, was scored according to CTCAE version 3.0. Tolerability was defined as the ability consume ≥ 75% of the intended number of muffins and the lack of dose-limiting toxicity, defined as ≥ grade 3 gastrointestinal toxicity. The desired tolerability rate was 80%.Fisher’s exact test was used to calculate whether there was a statistically significant difference between the tolerability and rates of pneumonitis and esophagitis. The Wilcoxon rank sum test was used to calculate whether there was a statistically significant difference between the percent of prescribed muffins ingested in the two arms.Fourteen patients were enrolled in this study and consumed at least one control (n=7) or one flaxseed muffin (n=7). Systemic oxidative stress can be estimated via urinary isoprostane measurements, normalized to urine creatinine. Panel A of p=0.56 by Fisher’s exact test). There were no cases of pneumonitis in the flaxseed arm compared to three in the control arm (p=0.19).There were no adverse effects directly related to administration of flaxseed-containing muffins. The median follow up was 9.9 months. Six of 7 (86%) patients died in each arm. Two-year overall survival was found to be 30% in both arms. Of the patients who died in the control arm, three patients had Distant Metastases (DM), two of whom had metastases on presentation as well, and one had a Local Recurrence (LR). In the flaxseed arm, there were three patients with DM (1 pre-existing DM) and 1 LR.We assessed the feasibility of incorporating flaxseed in the treatment of a small cohort of patients with advanced non-small cell lung cancer. Adherence to the flaxseed diet in the baked muffin formulation was below required levels for the study and therefore the trial was terminated after the first dose level. Here, we present the initial analysis of the patients enrolled. Markers of flaxseed metabolism, enterodione and enterolactone, were detected in the sera of the flaxseed group undergoing therapy. Urinary biomarkers of systemic oxidative stress (isoprostane and 8-oxo-D-guo) increased in response to radiotherapy. Trends toward reduced rates of pneumonitis were seen in FS-fed patients. More patients in the FS arm received concurrent chemotherapy than in the control arm; and despite this difference, pneumonitis rates were lower in the FS arm.Poor adherence to flaxseed supplementation in the muffin formulation was likely multi factorial. Patients noted intolerable taste and/or texture with flaxseed muffins, which is of prime importance in patients with anorexia and odynophagia due to chemoradiotherapy. Recently, novel formulations of flaxseed and routes of administration have been designed and may circumvent this issue such as employing ground whole grain flaxseed as a condiment, flavor enhancer, or thickening agent.The antioxidant mechanism of flaxseed is either by direct hydroxyl radical scavenging or by counteracting lipid peroxidation. Thompson et al.  in theirPrior work by our group (21) had shown dietary FS given before and after thoracic radiotherapy prevented radiation induced pneumonopathy in a murine model. Lungs were evaluated at 24 hours post-RT for acute markers of radiation-induced injury and the FS-fed mice demonstrated reduced expression of lung injury biomarkers . At three weeks, there was decreased cellular injury from RT as measured by malondialdehyde (MDA) levels. At four months, mice had decreased hydroxyproline content and fewer inflammatory cells in their bronchoalveolar fluid analyses indicating less late damage.Here we demonstrated a response in two urinary markers of systemic oxidative stress, isoprostane and and 8-oxo-dGuo. Biomarker levels increased from radiotherapy and ultimately decreased at one-month post treatment. This observation was consistent with the understanding that inflammation increases during the course of radiotherapy. As acute effects of radiation diminish and tumor size decreases, there are lower oxidative stress levels. We saw that there was an increase in 8-oxo-dGuo that did not normalize after cessation of radiotherapy. This could relate to ongoing DNA damage in tumor and normal cells persisting post-RT or in conjunction with chemotherapy or other antibiotic therapy. Isoprostane levels appeared to increase more in the flaxseed arm between week 1 and the end of RT; however, there was also a greater decrease between the ends of RT compared to week 1. Taken together, there was an overall decrease in isoprostane level from week 1 to the one month after RT. A dramatic decrease in isoprostane levels from baseline (one week prior to RT and FS treatment) occurred with initiation of flaxseed muffins. this study As seen in Given that this is a pilot study and therefore would only have adequate power to identify large differences between groups, no definitive conclusions can be made regarding the difference in oxidative stress levels between the flaxseed and control groups. There were no cases of pneumonitis in the flaxseed arm, which was encouraging. In addition, there were fewer cases of radiation induced esophagitis. Again, these were very important findings but need to be assessed in a randomized study with adequate power. In addition, as previously noted, chemoradiation is a large cause of gastrointestinal toxicity and therefore it is unclear how much of the intolerability of the flaxseed muffins may be from the chemoradiation itself. In addition, it should be noted that there appears to be an imbalance in the type of chemotherapy received by each arm of the study. However, due to the small number of patients, we are not able to statistically evaluate for this difference.Interpretation of these urinary biomarkers normalized to creatinine may be a source of error because of variable creatinine. With larger sample sizes, the effects of 8-oxo-dGuo and creatinine could be assessed separately in multivariable regression models .in vivo [Studies by our group in vivo  have revin vivo . Additioin vivo .Flaxseed administration in the muffin formulation was not feasible due to a range of factors including patient anorexia, odynophagia and tolerability of the flaxseed muffin formulation. However, quantification of flaxseed bioavailability and urinary markers of systemic oxidative stress could be achieved. There was a trend towards decreased rates of pneumonitis in FS versus control (0% vs. 42.9%). Dietary flaxseed may be clinically useful as an agent to increase the therapeutic index of thoracic RT by increasing the radiation tolerance of organs at risk including the esophagus and lungs. Based on our preliminary data, further clinical trials investigating different formulations of flaxseed which are now available are warranted.