The biological mechanisms in the association between the metabolic syndrome (MS) and various biomarkers, such as 25-hydroxyvitamin D (vit D) and magnesium, are not fully understood. Several of the proposed predictors of MS are also possible predictors of parathyroid hormone (PTH). We aimed to explore whether PTH is a possible mediator between MS and various possible explanatory variables in morbidly obese patients.Fasting serum levels of PTH, vit D and magnesium were assessed in a cross-sectional study of 1,017 consecutive morbidly obese patients (68% women). Dependencies between MS and a total of seven possible explanatory variables as suggested in the literature, including PTH, vit D and magnesium, were specified in a path diagram, including both direct and indirect effects. Possible gender differences were also included. Effects were estimated using Bayesian path analysis, a multivariable regression technique, and expressed using standardized regression coefficients.Sixty-eight percent of the patients had MS. In addition to type 2 diabetes and age, both PTH and serum phosphate had significant direct effects on MS; 0.36  ) and 0.28 , respectively. However, due to significant gender differences, an increase in either PTH or phosphate corresponded to an increased OR for MS in women only. All proposed predictors of MS had significant direct effects on PTH, with vit D and phosphate the strongest; -0.27  and -0.26 , respectively. Though neither vit D nor magnesium had significant direct effects on MS, for women they both affected MS indirectly, due to the strong direct effect of PTH on MS. For phosphate, the indirect effect on MS, mediated through serum calcium and PTH, had opposite sign than the direct effect, resulting in the total effect on MS being somewhat attenuated compared to the direct effect only.Our results indicate that for women PTH is a plausible mediator in the association between MS and a range of explanatory variables, including vit D, magnesium and phosphate. The metabolic syndrome (MS) is a clustering of risk factors including abdominal obesity, insulin resistance and elevated blood pressure , and it Age and diabetes are well-known to be associated with MS . A numbeEstablishing the role of PTH in relation to MS is complicated by the fact that several of the proposed predictors of MS are also known to be associated with PTH. The synthesis and secretion of PTH is tightly regulated by serum levels of calcium and phosphate, but also serum concentrations of vit D and magnesium influence PTH levels ,20, and The mechanisms explaining the association between MS and various biomarkers are not well understood, and focusing solely on direct effects might overlook clinically interesting indirect effects. The above references indicate that PTH might be a mediator for MS, highlighting the need for analytical approaches that include both direct and indirect effects.The aim of this study was to explore whether PTH might have a mediating role in the association between MS and a series of possible explanatory variables in morbidly obese patients. Dependencies between MS and a series of variables suggested in the literature were depicted in a path diagram. Effect sizes were estimated by path analysis, a regression technique where several multiple regression models are combined, allowing variables to be both an outcome and an explanatory variable in the same model .th, 2005 and September 16th, 2008, at the Morbid Obesity Centre in the county of Vestfold, Norway. The study was approved by the Regional Committee for Medical Research Ethics (S-05175) and was performed in accordance with the Declaration of Helsinki [Individuals having BMI ≥40 kg/m², or a BMI ≥35 kg/m² with at least one obesity-related comorbidity, are defined as being morbidly obese. The data set used for the present analysis was a cross-sectional study of 1,017 morbidly obese Caucasian individuals, collected prospectively between November 28Helsinki .MS was diagnosed in patients with at least three of the following characteristics ; elevateThe data has been published previously, and further details on definitions, physical examination and laboratory analyses can be found elsewhere .Based on previous research the following seven explanatory variables were included in a statistical model for the occurrence of MS; age, type 2 diabetes (T2DM), PTH, vit D, calcium, phosphate and magnesium. The relations between the variables, as suggested in the literature, are summarized in a path model Figure . The effIt is well known that age is associated with an increased odds of MS and T2DM, while T2DM is associated with increased risk of MS ,17. Low PTH has been shown to be associated with increased odds for MS, even after adjustment for vit D . The synArrows in a path diagram represent suggested dependencies between variables, and absence of an arrow between two variables indicates that these variables are considered statistically independent in the model. All hypothesized direct and indirect relations among measured variables can be read off the path diagram.Descriptive statistics are presented as mean and standard deviation (SD) or frequency and percentage (%). Chi square and independent samples T-tests were used for univariate comparisons.The parameters of our proposed path model Figure  were estThe path diagram formed the basis for the path analysis, in which we obtain direct and indirect effect estimates simultaneously by combining four regression equations, each with one of the following variables as the outcome: MS, PTH, calcium and T2DM. In order to assess the validity of the underlying assumption of linearity in the separate linear and logistic regressions, generalized additive models (GAM)  were fitWe also wanted to test for gender differences. As a preliminary analysis, we therefore fitted the path model separately for men and women. This analysis suggested possible gender differences for several paths, and parameters for such possible gender effects were included in the full model and estimated.All continuous variables were standardized prior to the analysis. Standardization allows one to compare the relative importance of various effects in a model, whereas using original variables allows for comparison across studies . We mainFor pathways consisting of only continuous variables, e.g. calcium→PTH→MS, indirect effects can be found by multiplication of the regression coefficients along this pathway, while total effects can be found by summing effects along each separate pathway connecting two variables, e.g. calcium→MS plus calcium→PTH→MS. When binary outcomes are involved somewhere along a pathway, the situation is less straightforward; regression coefficients have different interpretations in linear and logistic regression, and merely adding and multiplying regression coefficients is not meaningful.Statistical analyses were performed in R 2.11 . The patOn the variables in our path model we had complete observations for 971 (95.5%) patients, which were included in the statistical analyses. Clinical and biochemical characteristics are summarized in Table We first performed a path analysis of the model in Figure Besides diabetes and age, only PTH and phosphate had significant direct effects on MS. The effect of T2DM was by far the strongest, while the estimated direct effect of PTH was 0.36 . This value implies that a 1 SD increase in PTH (2.3 pmol/l) results in an increase of 0.36 in the log odds of MS. Due to a significant gender difference, this direct effect of PTH on MS was significant for women only.Direct effects of age and phosphate were 0.31  and 0.28 , respectively, implying that an increase in 1 SD in age or phosphate  results in an increase in the log OR of MS by approximately one third. Due to significant gender differences, these direct effects on MS were also only significant for women.Even though the 95% CrI for the gender difference for age on MS included zero, removing this gender effect from the model actually resulted in a model with an increased DIC, arguing for keeping it in the model.All proposed explanatory variables of MS had significant direct effects on PTH. As a consequence, they all have significant indirect effects on MS for women. The strongest direct effects on PTH were vit D and phosphate; -0.27  and -0.26 , respectively. This value implies that an increase of 1 SD in vit D or phosphate  corresponds to a mean decrease of approximately one fourth SD in PTH (-0.58 pmol/l). However, phosphate also has an indirect effect on PTH, mediated through calcium. The direct effect of phosphate on calcium is 0.27 , while the direct effect of calcium on PTH is -0.09 . The total effect of phosphate on PTH is thus -0.26+(-0.09)*0.27 = -0.28; about a 10% increase compared to the direct effect alone. While the effect of phosphate on PTH, and in result MS, is partly mediated through calcium, the effect of magnesium on MS is mediated through both PTH and T2DM. Due to T2DM being a binary outcome along one of the pathways, this total effect cannot be quantified.The direct effect of age on PTH was 0.20 . For the direct effect of age on PTH there was also a significant gender effect, resulting in this direct effect of age being significant for women only. For women, the total effect of age on MS is thus split into a direct path and two indirect paths; one via T2DM and one via PTH.A summary of the statistically significant paths for men and women is presented in Figure Previous reports have addressed the effect of PTH on MS, and discussed whether the variable should be adjusted for when estimating the effect of other possible predictors of MS -17. Our The use of path diagrams and analysis of structural models is a potentially valuable tool for increasing biological understanding , and is The crucial task in path analysis is to formulate a plausible path diagram based on existing evidence and current biological concepts. Path analysis is a multivariable regression technique and the hypothesized directions of arrows in the path diagram will thus affect results: Changing the direction of an arrow changes a variable's status from explanatory to outcome, or vice versa.We recognize that while more complex than traditional multiple regression models, our path model is still only one of many plausible models that can be hypothesized in order to explain the underlying biological mechanisms. There are several possible variables not included in our model that might influence estimates and considerations of direct and indirect effects, for example creatinine . Both alThe hypothesized mediating role of PTH in relation to MS was partly confirmed in our data. Of the included explanatory variables, several did not have significant direct paths to MS, while all but one had significant direct paths to PTH, implying significant indirect effects on MS. There was, however, a significant gender difference in the direct path from PTH to MS, implying that this direct effect was only significant for women. The proposed mediating role of PTH is as such a possible property for women only.The gender specific association between PTH and MS might be explained by some of the properties of the patient population. Men have a higher prevalence of diabetes than women (32% vs 22%) and MS (73% vs 65%) Table . The diaThe increased effect of age on PTH was also only significant for women. For men there was no direct effect on MS of neither PTH nor age. In other studies, increasing PTH levels seem to be associated with MS in older men, but not in women and younger men ,13,42. TWeight reduction and higher intakes of calcium and vitamin D have been found to be associated with decreases in PTH levels ,29. So eExactly why lowered PTH values are associated with reduced odds of MS is unclear. A recently published link between PTH and MS in older men was explained by insulin resistance, high blood pressure, hyperglycemia and low HDL-cholesterol ,13. The Whether lowering of serum PTH does indeed translate into beneficial effects on MS remains unclear, and cannot be assessed in a cross-sectional study; whether PTH is a mediator in the biological processes cannot be confirmed by a study like ours. We want to emphasize the need for experimental studies to assess the molecular mechanisms, as well as the importance of interpreting results from observational studies with caution concerning causality. However, with PTH regulative medications like cinacalcet  on the market, a randomized controlled trial can be carried out.Lower levels of phosphate have been shown to be associated with the components of MS , and areLow serum magnesium has been shown to be associated with T2DM and MS ,17,24,25Bayesian methods has only been used to a limited extent in clinical research, but the WinBUGS software has made Bayesian methods more easily available . While tThe current study is based on a relatively large, homogenous sample of morbidly obese individuals, which is further strengthened by the prospective collection and registration of data. In our model all variables are effectively adjusted for one another, and all parameters estimated simultaneously, reducing the effect of multiple testing.The study had some limitations. First, the cross-sectional design makes it impossible to establish cause-effect relationships. Though potentially graphically convincing and appealing, a path model cannot itself assess causality. The directions of arrows in the path diagram are a crucial point. For this, micro-level biochemical experiments are needed. Second, the results may not be valid in non-white populations or among non-obese subjects. Finally, we cannot exclude the possibility that referral of patients to a tertiary care center might have introduced a selection bias.The results of our study, combining current biological concepts and empirical data, suggest that PTH is a plausible mediator in the association between MS and various demographic variables and biomarkers, including vit D, phosphate and magnesium, in morbidly obese individuals. Due to significant gender differences, in our data this mediating role was only significant for women. Mechanisms like metabolic pathways are complex, and graphical models like the current can be useful tools.The authors declare that they have no competing interests.JR contributed to the conception and design, the statistical analyses, interpretation of data, drafted the manuscript and revised it critically for important intellectual content. BvC contributed to the concept and design, statistical analyses, was involved in drafting the manuscript and revised it critically for important intellectual content. JH contributed to the acquisition of data, statistical analysis and interpretation of data, drafted the manuscript and revised it critically for important intellectual content. All authors read and approved the final manuscript.R2WinBUGS[All path estimates presented in this paper were calculated using Markov Chain Monte Carlo simulation (MCMC) in WinBugs , run thrR2WinBUGS in the fR2WinBUGS. StochasR2WinBUGS. We usedR2WinBUGS, but theR2WinBUGS.


Certain types of the Human Papillomavirus (HPV) are sexually transmitted and highly associated with development of cervical dysplasia and cervical cancer but the distribution of HPV infection in the North, particularly amongst First Nations, Metis, and Inuit peoples, is little known. The purposes of the study are to identify the prevalence of type-specific HPV infections and the association of different HPV types with cervical dysplasia among women in Northern Canada.This was a cross-sectional study with attendants of the routine or scheduled Pap testing program in the Northwest Territories (NWT), Nunavut, Labrador and Yukon, Canada. Approximately half of each sample was used for Pap test and the remaining was used for HPV genotyping using a Luminex-based method. Pap test results, HPV types, and demographic information were linked for analyses.Results from 14,598 specimens showed that HPV infection was approximately 50% higher among the Aboriginal than the non-Aboriginal population (27.6% vs. 18.5%). Although the most common HPV type detected was HPV 16 across region, the prevalence of other high risk HPV types was different. The age-specific HPV prevalence among Aboriginal showed a ‘U’ shape which contrasted to non-Aboriginal. The association of HPV infection with cervical dysplasia was similar in both Aboriginal and non-Aboriginal populations.The HPV prevalence was higher in Northern Canada than in other Areas in Canada. The prevalence showed a higher rate of other high risk HPV infections but no difference of HPV 16/18 infections among Aboriginal in comparison with non-Aboriginal women. This study provides baseline information on HPV prevalence that may assist in surveillance and evaluation systems to track and assess HPV vaccine programs. Cervical cancer is the second most common cancer of the women worldwide. In Canada invasive cervical cancer and associated mortality among Aboriginal women have been 2–4 times higher than among non-Aboriginal women -4. From Human Papillomavirus (HPV) is a common sexually transmitted infection worldwide. Persistent infection with specific types of the virus is necessary for the development of invasive cervical cancer and its precursor lesions . HPV is Only very limited regional data ,3,15 proThe purpose of this study is to measure the prevalence of HPV infections by type and region and to determine their impacts on cervical dysplasia across Northern Canada.This was a cross-sectional study including all women aged 14 years or older who presented for a regular Pap test in the four regions of Northern Canada: NWT , Nunavut , Labrador , and Yukon . Women with a cervical cancer history and those who chose to opt-out were excluded from the study. Only the first sample collected was kept for analysis for women with more than one Pap smear performed over the study period. An opt-in consent process was used in Yukon which was different with the other jurisdictions. Given the universal coverage of the health care system in Canada there are no other Pap test providers in these areas.This research received ethical approval from the Health Canada Ethics Review Board, McGill University Ethics Review, and the local health authorities including NWT Department of Health and Social Services, Stanton Territorial Health Authority, the Yukon Department of Health and Social Services, and the Yukon Scientists and Explorers Act License. A Northern research licence was also obtained from Aurora Research Institute in the NWT. The project was facilitated by four data collection sites and the manuscript was reviewed by community stakeholders before being submitted for publication.Pap test screening is performed in each Territory and the Labrador health region in Canada. It is offered to all women aged 18–69 and young women aged 14–18 who wish to participate. Pap smears are collected at community health centres, medical clinics, or hospitals whichever appropriate. Pap test registration form is used to assemble personal record and demographic information on site. Demographic details including Aboriginal status, age, and region information were abstracted from the routine Pap test registration form. Aboriginal status was only available among NWT, Nunavut, and Yukon datasets. Only area code  was available in the Labrador dataset. Data were collected, organised, and owned by the local health authorities.Samples from NWT and Nunavut were collected in liquid based cytology (LBC) medium (SurePath®) and were shipped to the DynaLife Medical Laboratory in Edmonton, where cytology was read and remaining half of the specimen was kept for HPV testing. Samples from Labrador were also collected in SurePath LBC medium and shipped to Newfoundland Public Health Laboratory for Pap test interpretation. In Yukon, conventional Pap smear slides were taken and shipped to the Cervical Cancer Screening Laboratory, BC Cancer Control Agency. A sample for HPV testing was prepared by taking a second cervical sample and by rinsing the brushes in LBC medium. Samples for HPV genotyping were shipped from all four areas to the National Microbiology Laboratory (NML) in Winnipeg, Canada. Cervical cytology and HPV testing were done independently to eliminate the potential for reporter bias.For HPV genotyping an “in house” Luminex assay  developeCytological reports were issued according to the Bethesda system ,21. The Demographic information, HPV types and Pap test results were linked for HPV prevalence analysis. In the linkage dataset, 764 records (approximately 5% of collected data in the study) with one or more items of missing value were excluded from the analysis; 485 missing in age or age-group, 327 in ethnic status or ethnic area, and 399 in Pap test including 126 unsatisfactory cytology results.HPV_s and HR_s were defined as cases with only a single type of HPV infection and cases with only a single type of high risk HPV infection, respectively. Similarly, HPV_m and HR_m were defined as samples with two or more types of HPV or HR HPV infection. Samples with two or more HPV types were counted in for each type in type-specific analysis.Prevalence rates for type-specific HPV infection and cervical dysplasia were computed. Based on the 2008 census data among women in the NWT region  as the sA multivariate logistic regression analysis was used to explore the associations between HPV infection and cervical dysplasia, adjusted for Aboriginal status, age, and region. Odd Ratios (ORs) were calculated by different level: (a) infection with any HPV type, single HR infection, multiple HR infection, single LR infection, multiple LR infection, and infection with both LR and HR types, relative to no HPV infection, and (b) infection with both HR and LR HPV types relative to a single LR HPV infection.The proportion of cervical dysplasia caused by HPV infection in Northern Canada was measured by the exposure attributable risk fraction (ARF) and the population attributable risk fraction (PAR). In calculating the PAR, to avoid duplicate counting of those infected with both HR and LR, the exposed population was first stratified into those infected with only HR, only LR, and both. The PARs were summed across the strata. ORs are used in the calculation of both ARF and PAR: ARF = (OR-1)/OR and PAR = (Number of exposed cases x ARF)/total cases in the population. Furthermore, PAR of HR HPV infection = (No. of HR_s cases x ARF reference to HPV negative + No. of only multiple HR cases x ARF reference to HPV negative + No. of both HR and LR infected cases x ARF reference to LR HPV) / total cases .All data management and analyses were carried out using Statistical Analysis System 9.1. Two-tailed p value <0.05 was the criteria to determine the statistical significant .A total of 14598 cytology samples were collected and tested from NWT (n=6940), Nunavut (n=4683), Labrador (n=1370), and the Yukon (n=1605). In NWT and Nunavut the study covered more than 95% of the Program targeted population, respectively; but only 77% and 27% in Labrador and the Yukon in the study period. In this study, approximately 42% of women were non-Aboriginal and 58% were Aboriginal. The overall mean and median ages of participants were 35 and 32, respectively.HPV-DNA was detected in 26% of subjects Table . After wThe age specific HPV prevalence rate ranged between 13% and 48% Figure . The higIn Table Overall, 4.0% of Pap test results were abnormal (HSIL and LSIL); 4.7% in Aboriginal and 3.1% in non-Aboriginal group  who attended routine or scheduled pap screening over an approximately one year time period.The prevalence of overall HPV infection was 25.2% which was higher than in most countries -27 but sSimilar to most European and Canadian studies the two most common HPV types detected in Northern Canada were HPV-16 and 31 ,32-36. TThe prevalence analysis shows that HPV prevalence was highest among women aged less than 20 years, which is consistent with other studies ,31,38,39Noteworthy is that, despite the difference in prevalence of HPV types and cytological abnormalities between Aboriginal and Non-Aboriginal women, the proportion of HPV 16/18 in high grade lesion is about 50% in both groups. This is very similar to the average proportion of HPV 16/18 in HSIL in North America and worldwide . Since HCurrently routine Pap testing is one of the pillars to prevent cervical cancer. Other studies have found similar Pap test abnormality rates ,30. HPV Among participants with a normal pap test result, 26% of any type HPV infection among the Aboriginal group and 15% among the non-Aboriginal women Table  translatThere are potential limitations in the study. Firstly, no PCR protocol is ideal for all types of HPV detection. Any of the above comparisons in prevalence rates must be taken with caution as we have used the most recent IARC classification for HR HPV and a novel (Luminex) detection method as sensitive as Linear Array but able to detect more types than other previously reported technologies such as GP5+/6+, Elisa, or Hybrid Capture ,18. SecoIn Northern Canada, the prevalence of HPV infection was higher than other Canadian areas and varied by region. Between the Aboriginal and the non-Aboriginal group, the age specific distribution rates of HPV infections appear to be different; however, the effect of HPV infection to cervical dysplasia was similar. The relatively high prevalence of HR-HPV other than HPV 16/18, in particular among the Aboriginal group, call for further study to understand the impact of specific predictors . This stAAPR: Age-adjusted prevalence rate; ASC-H: Atypical squamous cells where HSIL could not be excluded; ASC-US: Atypical squamous cells of undetermined significance; ARF: Attributable risk fraction; CI: Confidence interval; HPV: Human papillomavirus; HPV_m: Two or more types of HPV infection; HPV_s: a Single type of HPV infection; HR: High risk; HR_m: Two or more types of high risk infection; HR_s: a Single type of high risk infection; HSIL: High-grade squamous intraepithelial lesion; LR: Low risk; LSIL: Low-grade squamous intraepithelial lesion; NML: National microbiology laboratory; NWT: Northwest territories; OR: Odds ratio; Pap: Papanicolaou test; PAR: Population attributable risk fraction; PCR: Polymerase chain reaction.There is no conflict of interest related to this work from any author.YJ: linked the datasets, analyzed the data, drafted the manuscript. PB: participated in the data collection, data analysis and results interpretation. AS: carried out all HPV DNA typing tests, revised the manuscript and helped to finalize the manuscript. YM: designed the study and made the substantial contributions to interpretation of data. YAL: involved in the majority of the project management work for NWT and Yukon. JAL: involved in data interpretation and helped revise the manuscript. SC: carried out the data collection in NWT and involved in the results discussion. AC: carried out the study in NWT site and involved in the results discussion. KK: carried out the study in NWT site and involved in the data collection and linkage. BH: carried out the data collection in Yukon site and provided with the critical review of the results. IS: initiated and managed the data collection in Nunavut. MA-R: initiated and carried out the data collection in Labrador-Grenfell Health, and involved in the results discussion. GJ: carried out Pap test for samples from NWT and Nunavut. JL: carried out Pap test for samples from Yukon. SR: carried out Pap test for samples from Labrador and involved in the results discussion. TW: involved in the data collection in NWT, Nunavut, and Labrador. AD: participated in the analysis of data and the critical review of the results. GJ: participated in conducting HPV type-specific surveillance and linkages to cytology results in Nunavut, involved in data interpretation and helped revise the manuscript. ST: involved in data interpretation and helped revise the manuscript. HM: performed the study and revised the manuscript. All authors read and approved the final manuscript.


We describe the treatment of a 46-year-old Saudi man with advanced stage liver metastatic neuroendocrine rectal cancer. The patient presented with a large liver lesion and rectal bleeding. He was cachectic, with a firm tender mass 20 mm above the anal verge. Computed tomography (CT) showed a mass 9.5 × 13 cm in size in the right hemi-liver, abutting the middle hepatic vein. The patient refused treatment, and consulted another hospital. After 3 months, he presented with the same symptoms in addition to delirium. Colonoscopy showed an ulcerating anorectal mass, from which a biopsy was taken. Repeat CT showed an increase in the size of the liver lesion to 17 cm and no change in the pelvis. The final histopathology report identified anaplastic small cell carcinoma. The patient underwent extended right liver resection followed by abdominoperineal resection, then 13 cycles of chemotherapy and monthly somatostatin injections. At the most recent follow-up, the patient had been disease-free for 48 months. Surgical resection (R0) of the primary and secondary tumor, followed by platinum-based chemotherapy can result in good survival in cases of small cell carcinoma with large liver metastasis, irrespective of whether the primary or secondary tumor is resected first. Neuroendocrine tumors include a wide spectrum of lesions including microcarcinoid, carcinoid, and mixed endocrine/exocrine tumors, and small cell carcinoma (SCC). SCC most commonly originates in the lungs, but it can originate anywhere in the body, including the esophagus , large bGastrointestinal (GI) SCC is a type of extrapulmonary carcinoma, and the GI tract is known to have the largest population of neuroendocrine cells . ApproxiStaging  and manaUnfortunately, patients with SCC of the GI tract have a dismal prognosis, with a median survival of only 6 to 20 months ,13. MoreWe therefore describe the first such case, to our knowledge, of successful surgical treatment of SCC of the rectum with advanced stage liver metastasis, followed by combination chemotherapy achieving long-term survival.A 46-year-old Saudi man was referred to Al-Hada Military Hospital, Taif, Saudi Arabia; he reported bleeding from the rectum for 6 months, along with decreased appetite, weight loss, and pain in the right upper quadrant of his abdomen. On examination, he was conscious and cachectic, with a mildly distended abdomen and a large hepatomegaly. Upon digital rectal examination, a firm and tender mass was found, 2 cm above the anal verge.Laboratory tests including complete blood count, renal panel, liver function test, carcinoembryonic antigen, and α-feto-protein were all normal. A CT scan of the abdomen showed a huge mass (13.5 × 8 cm) in the right liver reaching down to the right iliac fossa and pushing the abdominal viscera away.Unfortunately, the patient refused to undergo colonoscopy, and was discharged against medical advice, because he wished to get a second opinion. Three months later, he returned to our hospital. He presented to the emergency department with delirium and impaired liver function , alkaline phosphatase 203 μ/l 40 to 150 μ/l), aspartate aminotransferase 598 μ/l (5 to 35 μ/l), and alanine aminotransferase 373 (0 to 55 μ/l])). A repeat CT scan showed a dramatic increase in the size of the liver mass  (N0) , which was followed by monthly treatment with intrasmuscular somatostatin 20 mg, which is continuing. Positron emission tomography in May 2012 and CT in September 2012 did not show signs of recurrence. To date, the patient has been disease-free for 48 months.et al. [et al. reported a median overall survival of 15.3 months in a retrospective study of 24 patients [Only a few cases of patients with limited rectal SCC with long-term survival have been published. While only three cases -16 with et al.  reportedpatients . In the et al. [et al. [Only three cases of SCC of the rectum with liver metastasis have been published, and the patients were not treated surgically in any of these cases. In a report by Robidoux et al. , a patie [et al.  reported [et al. , in whicAfter reviewing the literature, we decided to attempt surgery on our patient because we could find no apparent significant advantages of chemotherapy or a combination of chemotherapy and radiotherapy in such cases (extensive disease), although this was based only on the three existing cases reported in the literature. Our treatment strategy was unique in that we started with the secondary tumor because the patient presented with deterioration of liver function, recent onset of delirium, and significant rapid growth of the liver with no change in the rectal tumor during the 3 months that elapsed between the patient deciding to seek another opinion and the time he re-presented to us. Surgery was followed by the established chemotherapy protocol (cisplatin and etoposide) to achieve the maximum result, followed by somatostatin therapy. This case is the first in which the patient had a huge liver metastasis of rectal SCC, and was successfully treated with surgery followed by chemotherapy. This treatment has so far resulted in long-term disease-free survival (48 months).Surgical resection (R0) of rectal SCC (limited or extensive disease), starting with the primary or secondary tumor, depending on the patient’s presentation, followed by (platinum-based) chemotherapy may result in better outcome and survival.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.BOA-J was the primary treating surgeon, followed up the patient, and wrote the paper. OAM: collected all the data, helped in writing the paper, and followed up the patient in the ward during his treatment process. All authors read and approved the final manuscript.


Background: Medically intractable chronic migraine (CM) is a                    disabling illness characterized by headache ≥15 days per month.Methods: A multicenter, randomized, blinded, controlled                    feasibility study was conducted to obtain preliminary safety and efficacy data                    on occipital nerve stimulation (ONS) in CM. Eligible subjects received an                    occipital nerve block, and responders were randomized to adjustable stimulation                    (AS), preset stimulation (PS) or medical management (MM) groups.Results: Seventy-five of 110 subjects were assigned to a                    treatment group; complete diary data were available for 66. A responder was                    defined as a subject who achieved a 50% or greater reduction in number                    of headache days per month or a three-point or greater reduction in average                    overall pain intensity compared with baseline. Three-month responder rates were                    39% for AS, 6% for PS and 0% for MM. No unanticipated                    adverse device events occurred. Lead migration occurred in 12 of 51                    (24%) subjects.Conclusion: The results of this feasibility study offer promise                    and should prompt further controlled studies of ONS in CM. Migraine is ranked by the World Health Organization as among the world’s most                disabling medical conditions , affectiIn 1999 Weiner and Reed reported the beneficial effects of subcutaneous occipital                nerve stimulation (ONS) in 12 of 13 patients who they believed to have occipital                neuralgia . Leads wPublished reports from open-label studies have demonstrated possible efficacy of ONS                in a variety of primary headache disorders, including CM ,9, clustThe study was prospective, multicenter, randomized, blinded, and placebo-controlled.                It was designed to obtain preliminary safety and efficacy data for ONS treatment of                CM. As a feasibility study, especially in a patient population that has been the                focus of very few randomized controlled trials, no primary endpoint was                prespecified; rather, a range of efficacy measures was identified and evaluated at                three months in comparison to baseline. Among the endpoints measured were reduction                in headache days per month, decrease in overall pain intensity                 and responder rate . A headache day was defined as each                day that a subject rated his or her overall headache pain intensity as ≥3.                CM was diagnosed using the second edition of the International Classification of                Headache Disorders (ICHD-II) . SubjectSubjects who met enrollment criteria were then randomized into one of three                    treatment groups, adjustable stimulation (AS), preset stimulation (PS) and                    medical management (MM), using a randomization ratio of 2:1:1, respectively.                    After implantation, the AS group was instructed to maintain the stimulator in                    the “on” position and to adjust the device to minimize pain.                    Serving as the control group for the AS group were subjects who received an                    implanted device that provided PS rather than AS. For these patients the device                    was set at a stimulation setting for one minute each day during the blinded                    phase of the study. A third group, also serving as a control group, received                    only MM during the blinded phase of the study. Unlike subjects in the AS and PS                    groups, who were required to maintain stable medication regimens , subjects in                    the MM group were able to adjust, change and optimize medication regimens as                    directed by their physicians. A fourth group, the ancillary group, met all entry                    criteria except response to occipital nerve block (ONB), which was an entry                    criterion for the other groups. A lack of response to ONB was defined as a                    failure to experience at least a 50% reduction in migraine pain within                    24 hours of the injection of 3–5 ml of 0.5% bupivacaine                    into each greater occipital nerve distribution. Patients in the ancillary group                    were implanted and allowed to adjust the stimulation and were treated                    identically to the AS group. The evaluation was conducted at seven centers in the US, one center in Canada,                    and one in the UK. The distributions of enrollment and treatment assignments by                    investigational center are shown in After confirmation of eligibility by the headache specialists, subjects were                    randomized into three treatment groups. Randomization was balanced across all                    centers but not within each center due to the anticipated relatively small                    number of subjects per center. The randomization was not stratified for baseline                    characteristics. A central randomization process provided and managed by                    Medtronic Neuromodulation (Medtronic) assigned a unique randomization code to                    each subject. Initially, randomization revealed only whether a subject was                    assigned to “medically managed” or “device                    implanted.” To maintain blinding in the device-implanted group, a sealed                    envelope with the complete randomization assignment (level of stimulation) was                    sent to implanter site personnel by Medtronic to be opened at the activation                    visit. Subjects were blinded to the anticipated value of adjustable stimulation                    over that of the preset stimulation. The sponsor’s study personnel                    (Medtronic) were not blinded to the randomized treatment assignments for                    individual subjects.Pulse amplitude: 0–10.5 VPulse rate: 3–130 HzPulse width: 60–450 µsThe study was conducted using the Medtronic model 7427 Synergy and model                    7427 V Synergy Versitrel implantable pulse generators, model 3487A                    Pisces Quad and model 3887 Pisces Quad-Compact leads, model 7489 and model 7482                    extensions, model 3550-25 accessory kit, model 3655-60 tunneling tool kit, model                    8840 clinician programmer, model 8870 application card, model 3628 dual-screen                    test stimulator and model 7435 Synergy EZ patient programmer. The product                    specifications of stimulation parameters for both models of implantable pulse                    generator are the same and as follows:. Subjects who did not complete the enrollment                        process, did not respond to ONB (after the ancillary group was filled) or                        who did not wish to continue had no further participation in the study.After approval of regulatory agencies, institutional review boards or ethics                        committees, subjects who provided written informed consent and met                        eligibility criteria were enrolled to the study. The enrollment process had                        three steps: preliminary enrollment visit, daily questionnaire enrollment                        phase and confirmation of enrollment visit. At the preliminary enrollment                        visit, information was collected, including health and well-being status,                        medical history and medication history. A preliminary diagnosis was also                        rendered. During the daily questionnaire enrollment phase, subjects                        completed an electronic daily questionnaire (EDQ) for a minimum of nine                        weeks, providing information about their headache, daily functional ability                        and medications taken. During the first week, subjects familiarized                        themselves with the questionnaire equipment. The purpose of the last eight                        weeks before the confirmation of enrollment visit was to establish baseline                        headache data. During the confirmation of enrollment visit, data from the                        EDQ was evaluated for diary compliance and headache features. Subjects who                        continued to meet eligibility requirements were given a short-acting ONB by                        the neurologist investigator. Those with a positive response to the ONB were                        eligible for randomization to the study. The first eight subjects study-wide                        who did not respond to ONB were entered into the ancillary                            groupSystem Manual to determine if a subject’s                        response to stimulation, as judged by adequate paresthesia coverage of the                        area of headache pain, was appropriate to receive a full implant. There was                        no trial of stimulation treatment. If during intraoperative testing the                        implanter believed inadequate paresthesia occurred over the location of pain                        based on the patient’s responses, the leads were removed, and the                        subject was followed for 30 days for safety and then terminated from the                        study. Subjects who felt adequate paresthesia over the target pain location                        during intraoperative testing continued with the implant procedure. Final                        lead placement was identified by X-ray. The implant procedure was performed                        with additional intravenous sedation to reduce patient discomfort; after                        lead placement was determined, the lead was locked in place using the twist                        connector, and a winged anchor was sutured in place. Connectors and                        extensions were used to allow placement of the neurostimulator just under                        the skin in the abdomen to reduce lead migration; if the abdominal site was                        determined to be inappropriate by the physician, the buttock was used. To                        further reduce the incidence of lead migration, the lead extension was                        placed with circular coils, creating strain-relief loops. Not all of these                        implant techniques, such as use of strain-relief loop, were employed                        consistently at the beginning of or during the study. However, a                        recommendation of using strain-relief loop and preference of abdominal to                        buttock implant location of the neurostimulator was provided to all                        implanters during the study when a number of lead migrations were                        reported.Using local anesthesia and fluoroscopic guidance, one or two leads were                        implanted subcutaneously, superficial to the fascia and muscle layer at the                        level of C1. Intraoperative testing was consistently performed according to                        the ONS The device was activated after the surgical site healed, between 7 and 14                        days after implantation. All subjects received ONS using parameters                        optimized by the physician on the basis of their response to treatment, but                        the duration of stimulation differed according to group assignment. Patients                        in the PS group received one minute per day of stimulation and were                        instructed that their neurostimulator had been pre-programmed to deliver the                        correct amount of stimulation as determined by their treating physician.                        Patients in both the AS and PS groups were not informed of the predicted                        effectiveness of their treatment. Patients in the AS and ancillary groups                        received a device programmer allowing them to turn the neurostimulator on                        and off and to make minor adjustments to settings, and they were instructed                        to maintain the stimulator in the “on” position as much as                        possible. Subjects in the PS group were not given a device programmer to                        adjust the settings.All enrolled subjects had follow-up visits at one and three months. Safety                        and efficacy were evaluated at the three-month visit. Subjects in the PS and                        MM (control) groups were offered adjustable ONS therapy after the                        three-month follow-up visit. Subjects in the ancillary group were treated in                        the same manner as AS subjects and followed the same visit schedule.                        Subjects in the AS, PS and AG groups who were on medications were required                        to maintain stable medicine regimens, although the frequency and dose of                        acute medications could change if necessary. Those in the MM group were able                        to adjust and change medication regimens throughout the three-month blinded                        phase, as directed by their physicians. After the three-month follow-up                        visit, all subjects, regardless of initial group, were able to adjust acute                        and prophylactic medication regimens as needed.The ONSTIM feasibility study has completed three-month blinded follow-up                        visits for safety and efficacy endpoints. Subsequent open-label, long-term                        follow-up visits included 6, 12, 24, and 36 months. Not all of these                        long-term follow-up visits have been completed at the time of this                        manuscript; hence, only three-month safety and efficacy results are reported                        here.The three-month safety objectives were evaluated by determining adverse                    device-related events (ADEs) for all implanted subjects, including ADEs reported                    during intraoperative testing and scheduled and unscheduled visits throughout                    the three-month follow-up period. ADEs were classified into those related to the                    components of the system, the implant procedure, device programming or device                    stimulation.Device Event Dictionary.Non–device-related adverse events also were collected for all subjects at                    scheduled and unscheduled visits beginning with the confirmation of enrollment                    visit and throughout the three-month follow-up period. They were classified                    using the MedDRA Version 8.0 dictionary and the Medtronic Neuromodulation                        Per protocol, there was no data safety monitoring board (DSMB) or clinical events                    committee (CEC) for this feasibility study. All adverse events regardless of                    device relatedness were reviewed and monitored by the Medtronic Neuromodulation                    medical advisor throughout the study. A serious unanticipated ADE or a                    percentage of subjects experiencing a specific serious ADE higher than                    previously reported were defined as criteria for consideration of modification                    or termination of the study.Data were collected using electronic diary for headache days, pain and duration                    measurements. Paper case report forms were used for data collection of safety                    and quality of life. Data collected for safety objectives included                    device-related and non–device-related adverse events; data collected for                    efficacy objectives included headache days, headache-free days, days with                    prolonged and severe headache, headache pain intensity, headache duration,                    responder to ONS therapy, functional impairment ,                    migraine disability assessment (MIDAS), quality of life (SF-36) and subject                    satisfaction.p < .05 as potentially informative,                    and these are nominally referred to as statistically significant throughout the                    paper, with actual p values not reported due to the exploratory                    nature of the analyses. Wilcoxon’s rank sum tests were used to analyze                    headache days, pain intensity, disability and quality-of-life outcomes; these                    summary data are presented as mean ± standard deviation.                    Fisher’s exact tests were used to analyze responder rate and subject                    satisfaction; these summary data are presented as frequency counts and                    percentages. For the safety objective of the study, descriptive summaries are                    presented. SAS software  was used for                    all data analyses.The sample size was chosen to gain experience with ONS therapy for the treatment                    of CM. In order to evaluate effectively the study design, a sample size of 24                    subjects in the AS group and 12 subjects in each of the PS and MM groups was                    required. In keeping with the exploratory nature of the study, it was not                    powered for a single primary endpoint. However, according to the protocol,                    statistical analysis was performed to allow more critical consideration of the                    data in order to identify factors and nuances that might be helpful in further                    studies. A per-protocol analysis, including all subjects who completed the                    electronic diary during the three-month blinded follow-up, was used to compare                    subjects who completed three months of stimulation therapy in the AS group with                    subjects in the PS, MM and ancillary groups who also completed three months of                    follow-up. Pairwise comparisons between the AS group and each of the three other                    groups were not adjusted for multiple comparisons and are presented only as a                    guide to interpreting the study. We considered differences with                        An interim analysis was conducted in January 2007 for business planning of future                    studies. No stopping rules were applied because the analysis was unrelated to                    safety, which is monitored and evaluated on an ongoing basis as described above.                    The results of this interim analysis were not provided to the investigators.The clinical investigation began on May 26, 2004, and the last subject was enrolled                on April 6, 2007. The final three-month follow-up visit was conducted on November                13, 2007. A total of 110 subjects aged 18 years and older were enrolled to achieve                the final sample size of 75 subjects assigned to a treatment group . Thirty-The subjects experienced migraine for an average of 22.0 years prior to the study                 and experienced CM headaches for an average of 10.0                years prior to study enrollment . The gender                ratio of males to females was approximately 1:4. Treatment groups were similar in                demographic and baseline headache characteristics . Table 3The overall outcomes comparing baseline observations and three-month data are                    presented in For the majority of outcome measures , the exploratory analyses showed no statistically                    significant improvement over baseline when comparing the AS group with the                    control groups (PS and MM), although a numerical advantage appeared to be                    associated with the AS group. Because the number of subjects in the ancillary                    group was small, reliable comparisons could not be made.The Profile of Moods States (POMS), MIDAS, SF-36, functional disability and                    subject satisfaction scores were also evaluated in this study. Exploratory                    analyses yielded significant differences between the AS and control groups in                    the measures reported here. The POMS was used to measure six areas of mood                    states: tension-anxiety, depression-dejection, anger-hostility, vigor-activity,                    fatigue-inertia and confusion-bewilderment. A lower score represents a decreased                    mood state. Reductions in POMS scores from baseline to three months were as                    follows: 8.7 ± 12.0 for AS,                    1.6 ± 10.1 for MM and 0.4 ± 9.4                    for PS. Sixty-six percent of subjects in the AS group and 25% of                    subjects in the MM group reported satisfaction with treatment at three months.                    Change from baseline in score on the functional disability scale was                    0.3 ± 0.5 for the AS group and                    0.0 ± 0.3 for the MM group. Change in acute medication                    use was 1.6 ± 7.6 in the AS group and                    −0.6 ± 5.0 in the MM group. Change in MIDAS                    average grade was 0.4 ± 0.8 for the AS group and                    0.0 ± 0.0 for the MM group, and change in MIDAS headache                    pain score was 1.3 ± 1.8 for the AS group and                    0.0 ± 0.9 for the MM group. Scores on the SF-36 Mental                    Health domain were 5.5 ± 9.7 and                    −1.5 ± 6.3 for the AS and MM groups,                    respectively.For the majority of outcome measures of disability and quality of life, including                    the functional disability scale, MIDAS scores and SF-36, the exploratory                    analyses showed no significant improvement over baseline when comparing the AS                    group with the control groups (PS and/or MM), although a numerical advantage                    appeared to be associated with AS in most cases. Because the number of subjects                    in the ancillary group was small, reliable comparisons could not be made.A total of 53 subjects underwent the implant procedure. Two subjects were                        intraoperative failures, leaving 51 subjects who were successfully                        implanted. Fifty-six ADEs occurred in 36 of the 51 subjects. Non–device-related adverse events involved principally worsening of                        migraine during the three-month testing period as compared with baseline.                        Nine percent of the AS group, 41% of the PS group and 24% of                        the MM group reported increased migraine. Adverse events related to                        medications were similar across treatment groups and ranged from 6%                        to 18%. The data from this feasibility study suggest ONS for medically intractable CM can be                carried out relatively safely and is worthy of further study for this indication.                Although the study was not prospectively powered for efficacy evaluation, the                39% responder rate in the AS group is comparable with response rates                achieved with widely used preventive CM treatments, such as topiramate ,17. MoreThe decision to employ ONS for the diagnosis of CM rather than for occipital                neuralgia is based on the clinical phenotyping of patients responding to therapy                    . This isAlthough the complete pathophysiology of CM remains unclear, a role for the                trigeminocervical complex seems established . The intThis study indicates that the adverse events from the period of implantation to three                months are not prohibitive to further exploration of therapy. The primary ADEs                involved lead migration or dislodgement and incision-site complications. Lead                migrations, although not generally defined as a serious adverse event, ultimately                require a repeat invasive procedure and therefore should not be minimized in                importance. There was no evidence of ADEs leading to long-term complications or                potential nerve damage. Additional studies are currently reviewing the long-term                effects of ONS. No unanticipated ADEs occurred in this study. Moreover, from this                study we have gained sufficient technical information to move toward reducing lead                migration in future studies, including considerations of appropriate strain-relief                loops, anchors, implant locations, implant procedures and lead migration assessment                techniques.Even if the design and power of this study were sufficient to provide statistically                reliable results, a responder rate of 39%, or any of the other positive                findings reported here, might not seem compelling in support of neurostimulation.                Nonetheless, from the perspective of an often treatment-refractory population of                patients, such a finding could represent an important therapeutic signal for at                least a subset of patients that justifies continued study in pursuit of ultimate                therapeutic value. Patients with CM are often left without effective treatment,                causing patients to lead lives that are painful and compromised. Patients with CM                often travel from physician to physician and center to center, and are prescribed                long lists of powerful medications, many in complex combinations that both                compromise function and impose risk. Were a well-designed, controlled, and blinded                study to demonstrate that even a small subset of patients with CM achieved reliable,                substantial, prolonged benefit from ONS, we believe it would represent an important                contribution to the care of these patients.This study had several limitations. First, trial duration was short. A longer period                of observation might reveal a different pattern of adverse events. Second, complete                patient and investigator blinding was difficult to achieve. Although subjects and                neurologists were blinded, implanters were necessarily unblinded for conduct of the                study. In addition, maintaining subjects in a blinded mode is difficult in any study                when stimulation can be perceived. The PS group did not have a device programmer,                which also could have led to unblinding. Third, the preset stimulation might have,                itself, had a therapeutic benefit, although this did not appear to be the case in                the current study. Finally, the parameter of headache days could have used more                sensitive definition, such as days with moderate or severe headache specified by not                only intensity but also duration.Many questions beyond basic efficacy remain unanswered for this therapy. Long-term                safety of stimulation, durability of any positive benefit and technical and                electronic reliability remain untested. Psychological and clinical eligibility for                stimulation, the effect of medications on stimulation response (or lack thereof),                interventional technique considerations and device and lead design factors have yet                to be determined or fully explored.There are many challenges to overcome before reliable conclusions on matters of                efficacy and safety of ONS in medically intractable CM can be established.                Developing a bona fide placebo group for surgical implant studies is particularly                important—and particularly difficult. The perception of paresthesia may be                required to obtain pain control, but technical issues make creation of this                sensation in a placebo group a major challenge for the design of randomized                controlled trials. Although the PS group represents a step to address this issue,                more must be done to assure that the placebo stimulus is not itself therapeutically                effective. In addition, better screening criteria are needed. However, because                medically intractable CM is a frequent cause of disability and a therapeutic                challenge in neurological practice, attempting to overcome these in future studies                is a worthwhile pursuit.On the basis of the current findings and in light of previously published work, we                believe further investigational pursuit to evaluate the efficacy and safety of ONS                for medically intractable CM is justified. Further study would be enhanced by                improved stimulator design, implanting technique and lead design and by a                well-targeted, carefully selected study population, more robust endpoints, longer                trial duration and improved blinding techniques. Reliable conclusions regarding                efficacy cannot be established on the basis of this study alone. Nonetheless, the                results of this feasibility study offer promise and should prompt further study of                ONS in medically intractable CM.The study was designed by the authors and employees of Medtronic Neuromodulation. The                full data set has been available to the authors, and data were analyzed by                Medtronic. The lead authors  wrote the first draft of the manuscript. All                content, interpretation, implications of results, discussion and conclusions were                composed by the authors.Neurologist, and research                support within the past two years from Medtronic, Advanced Neuromodulation Systems,                St. Jude Medical, the National Institute of Neurological Disorders and Stroke and                Mayo Clinic. SDS has consulted for Novartis and is on the speakers bureau of Endo                Pharmaceuticals, GSK and Merck. SDS has served on advisory panels of Medical                Corporation (AGA), Allergan, Capnia, Coherex, GSK, Lilly, MAP, Medtronic, Merck,                NuPathe and Pfizer, and has received research support from AGA, Advanced                Neuromodulation Systems, Allergan, Boston Scientific, Coherex, Endo Pharmaceuticals,                GSK, Lilly, MAP, Medtronic, Merck, NuPathe and Valeant Pharmaceuticals. SM is an                employee of Medtronic Neuromodulation and owns Medtronic stock through employment.                MS is an employee of Medtronic Neuromodulation and owns Medtronic stock through                employment. PJG has consulted for, and received honoraria from, ATI, Medtronic and                Boston Scientific in the area of neuromodulatory treatment of headache.JRS has received consulting/advising compensation for activities with Merck,                Ortho-McNeil-Janssen Pharmaceutical (OMP), Purdue Pharma, Neuralieve, Allergan,                Medtronic, Pfizer and St. Jude Medical. He holds stock and/or stock options in                Pozen. He receives research support from GlaxoSmithKline (GSK), Johnson &                Johnson, Eli Lilly, Merck, St. Jude Medical, Map Pharma, Nupathe, Zogenix, Neura                Axon and Boston Scientific. DWD has provided consulting services for the following                companies within the past two years: Allergan, Addex, Alexza, Coherex, Eli Lilly,                Endo, GSK, HS Lundbeck Kowa, MAP Pharmaceuticals (MAP), Medtronic, Merck, Minster,                Nautilus, Neuralieve, Neuraxon, Novartis, NuPathe, Pfizer and Zogenix. DWD has                received editorial honoraria from Wiley-Blackwell, the American College of                Physicians, SAGE Publications and the


Quantifying effects of toxicant exposure on metabolic processes is crucial to predicting microbial growth patterns in different environments. Mechanistic models, such as those based on Dynamic Energy Budget (DEB) theory, can link physiological processes to microbial growth.Pseudomonas aeruginosa optical density growth data for seven levels of cadmium exposure. The model reproduces growth patterns for all treatments with a single common parameter set, and bacterial growth for treatments of up to 150 mg(Cd)/L can be predicted reasonably well using parameters estimated from cadmium treatments of 20 mg(Cd)/L and lower.Here we expand the DEB framework to include explicit consideration of the role of reactive oxygen species (ROS). Extensions considered are: (i) additional terms in the equation for the “hazard rate” that quantifies mortality risk; (ii) a variable representing environmental degradation; (iii) a mechanistic description of toxic effects linked to increase in ROS production and aging acceleration, and to non-competitive inhibition of transport channels; (iv) a new representation of the “lag time” based on energy required for acclimation. We estimate model parameters using calibrated Our approach is an important step towards connecting levels of biological organization in ecotoxicology. The presented model reveals possible connections between processes that are not obvious from purely empirical considerations, enables validation and hypothesis testing by creating testable predictions, and identifies research required to further develop the theory. Investigations of the dependence of bacterial growth curves on exposure are often used to consider the ecological importance of toxicants. Although such dependence is an aggregate, population-level measure of all toxic effects, it is a consequence of processes at the cellular or molecular levels.Toxicants may affect the onset, rate, and extent of bacterial growth curves by directly increasing mortality, impacting nutrient uptake by affecting cross-membrane transport , and by Dynamic Energy Budget (DEB) theory provides a comprehensive framework for connecting molecular-level processes to individual physiology and population growth of all organisms , and hasPseudomonas aeruginosa growth  area of the bacteria and the outside concentration  as the main cause of production of damage-inducing compounds (e.g. DNA damage) and, consequently, aging and cell damage .We extend the formulation to include effects of exposure assuming that the toxicant produces ROS and, therefore, increases aging acceleration in proportion A repair , but theThe hazard rate, B theory , the hazpopulation of unicellular organisms which is, in its entirety, much more similar to a multicellular than an unicellular organism.The second term on the right-hand side of (5) represents dilution by growth  to cell death come from the ROS produced by the toxicants, and the resulting increase in aging acceleration. Therefore, any effect the toxicant may have on hazard is accounted for through increase in aging acceleration, and equation (5) captures the effect of toxicants on mortality.Toxic effects on mortality in previous DEB models are accounted for by adding to ntration . We, howntration . This imP. aeruginosa. Modeling the exact mechanism of environmental degradation is, however, beyond the scope of this paper. We therefore account for effects of environmental degradation in a general way.In addition to depleting nutrients, bacteria may degrade their environment in other ways. Given the large cell densities reached in bacterial cultures, the negative effects have the potential to be significant. Yeast, for example, degrade their environment by producing alcohol, and levels of dioxygen in post-exponential growth in plate cultures of aerobic bacteria have shown to be immeasurably small . In condet al. , even though aeration through the air/supernatant interface has not been accounted for. In microplates, as evidenced by dioxygen concentrations presented in Kocincova al. 2008 , aeratioOne of the simplest ways to model the decline in organism's ability to acquire and utilize energy due to environmental degradation is to exponentially reduce the parameter characterizing energy conductance . It also includes a novel connection between the aging acceleration and physiological rates that feature in DEB models. Our primary aim was to find a minimal mechanistic representation consistent with the results of one set of experiments , and to Development of the model reported here was preceeded by work on a much more complex model that, inter alia, distinguished “active” and “inactive” cells, and implemented toxic effects on additional physiological processes such as maintenance, costs of growth, and cell lysis. We first created and fitted a model based on one set of assumptions, then repeated the process with a slightly different set of assumptions until we reached a balance of model complexity and ability to capture main features of the data. The final model reached in this iterative process has a fair number of parameters, with which, if the model were wholly empirical, it might have been possible to fit practically any set of curves. However, the mechanistic nature of the model, and the need to satisfy eight curves at the same time with a single parameter set, severely limits the shape of the growth curves that can be obtained  for the DEB variables. Less obvious is the need for careful experimental design in studies intended for comparison of intracellular quantities such as ROS levels, for example the data shown in al. 2009 ), and thal. 2009 . If the al. 2009 , where dInvestigating ROS dynamics during the acclimation phase could provide insights into reasons behind the observed lag phase. Our assumption that all treatments require the same acclimation energy because they need to produce the same molecular machinery may be wrong if cells can adjust the magnitude of the response. For example, the lowest two concentration treatments give maximum cell densities significantly lower than the observed, partly because energy spent on acclimation reduced total energy available for growth. If lower exposure concentrations required less acclimation, the maximum cell density would have been higher. This would also reduce the underestimates of pre-exponential growth for low exposures.Our framework can be expanded to link actual molecular mechanisms of ROS creation and neutralization to the production of damage-inducing compounds (and increases in aging acceleration). Currently, we followed DEB theory in assuming a fairly simplistic dynamics of ROS: each ROS produced gives a fixed contribution to aging acceleration. We could instead differentiate between reactive species and their effect on aging acceleration to consider how different environmental conditions affect bacterial growth: certain reactive species could produce more damage-inducing compounds than others, while some reactive species could cascade through multiple forms before being neutralized, increasing aging acceleration with each transformation. Different toxicants could produce different numbers and species of ROS. The physical location of the ROS could also be taken into the account: damage to the membrane affects different processes than damage to the DNA. Available data and knowledge, however, simply do not allow such elaborations.Clearly, additional data are necessary to tune the current model into a reliable theoretical framework which would enable accurate predictions. Ideally, we would want data sets that allow for independent testing of model assumptions and reduce ambiguity in parameter estimation. Data that would have greatly helped in the present work include: media characterization throughout the experiment to determine possible mechanisms of environmental degradation, including a time series of dioxygen availability, supernatant ROS, and pH; time-series of cellular ROS and/or other reactive species for all or most treatments to help determine initial delay mechanisms, validate assumptions on accumulation of damage-inducing compounds, and help estimate parameters; time-series of cellular and supernatant dissolved cadmium concentrations to fine-tune the dynamics of bioaccumulation; alternative methods of characterizing population size; any and all physiological processes, such as respiration, that can be measured.et al.  measurements non-linearly related to cell concentration to a measure of cell concentration, and then convert that measure into C-moles.et al. , but thal. 2009  includedP. aeruginosa in Luria-Bertani broth were inoculated from frozen stock ; we were unable to obtain a reasonable fit after removing any one of the remaining assumptions. This, combined with a maintenance rate coefficient realistic for bacteria, gave us some confidence in the model; the fact that our predictions of aging acceleration reflected those expected from the observed ROS dynamics, and the ROS density measured by Priester al. 2009   should have much higher priority than further statistical refinement of the current analysis.Even though the least-squares method converged, there were a number of colinearities among parameter estimates. Furthermore, we cannot be certain that our routines did not locate a local minimum of the residual sum of squares. We investigated this by exploring the parameter space manually, but there remains the possibility that a better data fit than presented here exists. Finding it is, in our opinion, beyond the scope of the manuscript which showed that the model To minimize the possible influence that Cd salts had on optical density for low cell concentrations, we used the initial value of the structural volume of the control for all treatments. This decision recognized that all inocula came from the same stock. We arbitrarily assumed that cells had 50% of the maximum energy density when transferred from the inoculum by setting the initial energy density to 0.5. The precise value chosen did not affect our ability to fit the data. The initial substrate concentration was set to unity , and all other initial values  were set to zero.Figure S1Comparison of raw (left panel) and calibrated data (right panel).(TIF)Click here for additional data file.Figure S2Bioaccumulation for 5 (lowest solid line), 10, 20, 37.5, 75, 115, and 150 (highest solid line) mg/L total cadmium.(TIF)Click here for additional data file.Figure S3Predictions using alternative data sets for fitting. Data points marked with ‘x’: used in fitting; data marked ‘o’: not used in fitting. Solid line: simulations of data not used in fitting; dotted line: simulations of data used in fitting. Top left panel: predicting 5, 10, 20, 37.5 and 75 mg/L using 0, 115, and 150 mg/L. Top right panel: predicting 5, 10, 37.5, 115 and 150 mg/L using 0, 20 and 75 mg/L. Lower left panel: predicting 10, 20, 75, 115 and 150 mg/L using 0, 5, and 37.5 mg/L. Lower right panel: predicting 5, 20, 75, 115 and 150 mg/L using 0, 10 and 37.5 mg/L.(TIF)Click here for additional data file.Information S1Supporting Information. Additional results, overview of the basic DEB model and scaling, dicussion on how mortality affects the model equations, derivation of acclimation dynamics, and possible extensions of the model.(PDF)Click here for additional data file.


Takayasu arteritis (TA) is a chronic vasculitis of unknown origin, affecting mainly the aorta and its main branches. As a result of the inflammation, stenosis, occlusion or dilatation of the involved vessels may occur and cause a wide range of symptoms. It has been described in association with various auto-immune disorders (mainly inflammatory digestive tract diseases). However, only few cases of TA associated with sarcoidosis have been reported, raising the question of an association by chance.R).We report a case of a 34 year-old woman, with one year history of sarcoidosis, who presented with asymmetric high hypertension revealing inflammatory humeral, axillary and subclavian arteritis related to TA, successfully treated by steroid and immunosuppressive therapy(MethotrexateTA and sarcoidosis may be related, rising the hypothesis that TA or Takayasu arteritis-like granulomatous vasculitis may be, in fact, a complication of sarcoidosis. Takayasu arteritis (TA) is a chronic inflammatory disease of large arteries which progressively develop stenosis, occlusion or aneurismal degeneration. The clinical presentation is characterised by an acute phase with constitutional symptoms, followed, months or years later, by a chronic phase in which symptoms relate to fibrosis or occlusion of vessels. Angiography is the gold standard for diagnosis and for topographical classification and it correlates with symptoms and prognosis. Proinflammatory cytokines and, among these, tumor necrosis factor-alpha  are increased and play a pathogenetic role in the development of disease. Laboratory findings are non-specific. Treatment of active disease is primarily based on corticosteroids but other immunosuppressive drugs are frequently needed. Anti-platelets agents, statins and antihypertensive drugs are frequently considered .Sarcoidosis, too, is a systemic inflammatory disease which can affect virtually any organ system. The lungs are most commonly affected, but often there is also affection of the musculoskeletal system, the cutaneous and central nervous systems, and the heart. Histology shows granulomas consisting of a central zone with macrophages, epithelioid cells, and multinucleated giant cells in addition to activated CD4 lymphocytes, and a peripheral zone with macrophages, fibroblasts, and CD4 and CD8 lymphocytes. Inclusion bodies are commonly found. Vasculitis may be found occasionally in patients with sarcoidosis. It has not been confirmed, however, whether vasculitis is a true manifestation of the disease or an occasional, incidentally concurring abnormality.Concurrence of sarcoidosis and TA were reported in few cases.A 34 year old woman was admitted to the hospital with dyspnea, submandubular gland tumefaction and parotid swelling.On physical examination, we found the tumefaction of the parotid and the submandibular gland. The respiratory frequency was 24 cycles per minute. The rest of the clinical examination was without abnormalities.Laboratory results were as follows: Haemoglobin 10 g/dl, white blood cell count (WBC) 11 200/mm3, platelets 450 000/mm3, erythrocyte sedimentation rate (ESR) 76 mm/h, calcium 12 mg/dl . Muscle enzymes, ANA, rheumatoid factor, VDRL, and complement components C3 and C4 were negative. The angiotensin converting enzyme (ACE) level was 135 UI/L.Chest radiography showed "Bilateral interstitial involvement". Thoracoabdominal CT scan showed "Multiple mediastinal and retroperitoneal lymphadenopathy, with moderate hepatomegaly". The respiratory functional test showed the presence of a restrictive syndrome.The lung fibroscopy was normal. The biopsy confirmed the presence of a non caseating granuloma in the bronchial mucosa. The bronchoalveolar lavage showed a predominantly lymphocytic cell with the elevated Cd4/CD8 ratio.Ophthalmological examination confirmed the presence of dry eye syndrome. There was no uveitis. The salivary gland biopsy confirmed the presence of a granuloma without caseous necrosis.Sarcoidosis was diagnosed, and the patient received prednisolone 60 mg/day. After 1 month of treatment, all complaints have been resolved.Seven months later, when she received 10 mg/day of prednisolone, she consulted for respiratory distress. Her left brachial and radial pulses were absent, and her blood pressure was 180/120 mm Hg as measured from the left arm. Bruits were audible over the course of the left carotid artery and the left and the right subclavian artery. The patient had elevated ERS (100 mm/H).An Ultra Sound Colour Doppler (USCD) was performed showing a circumferential parietal swelling interesting the left subclavian artery reducing his superficies of 76%, demodulating the Doppler spectrum and edema of the vessl wallFigure . These dWe diagnosed the patient having TA associated with sarcoidosis. She received prednisolone 40 mg/day with Methotrexate 10 mg/week and amlodipine 5 mg/say.As a response to treatment, blood pressure was normal 2 weeks after. The USCD control confirmed the therapeutic benefice showing a reduction of the parietal anomalies.About a dozen cases of the association TA and sarcoidosis were reported in the literature. -6; some TA can be associated with Crohn's disease , RCH, syThe association between sarcoidosis and TA or other forms of aortitis may be rare. However, TA or similar forms of aortitis are too rare to indicate that the reported concurrence with sarcoidosis is merely governed by chance. In fact, all these patients had several common features: (a) usually sarcoidosis preceded TA; (b) the time between the diagnosis of sarcoidosis and that of TA was several years (eight or more) in most of the patients; (c) the aorta and/or its major branches was affected; (d) 50% of the patients had uveitis, which is much less common in frank sarcoidosis; and (e) all patients responded to glucocorticoid treatment.Association of TA and sarcoidosis raise a number of questions of a possible link between these 2 diseases. Vascular injury (or "granulomatous vasculitis), and especially the location in the aorta thoracic and/or its branches, could be a complication in the course of sarcoidosis -3,10. HoThe association between sarcoidosis and TA are rare but not accidental; complete vascular clinical examination should be performed in those patients in order to detect asymptomatic underlying inflammatory arteritis.The relatively low prevalence of sarcoidosis, and even more so the rarity of aortitis, suggests that this number is relatively high and too high to be a coincidence; can we say that sarcoidosis and TA appears to be 2 related diseases or that Takayasu arteritis or Takayasu arteritis-like granulomatous vasculitis may be, in fact, a complication of sarcoidosis?Informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.AH, RK, OH, OB and SM: Diagnosis the disease and monitoring the patient RS and MG: Have carried out the radiological explorations.All authors read and approved the final manuscript.