Although renal pathology is highly predictive of the disease course in lupus nephritis, it cannot be performed serially because of its invasive nature and associated morbidity. The goal of this study is to investigate whether urinary levels of CXC ligand 16 (CXCL16), monocyte chemotactic protein-1 (MCP-1) or vascular cell adhesion molecule-1 (VCAM-1) in patients with lupus nephritis are predictive of particular features of renal pathology in renal biopsies obtained on the day of urine procurement.CXCL16, MCP-1, and VCAM-1 levels were measured in urine samples from 74 lupus nephritis patients and 13 healthy volunteers. Of the patients enrolled, 24 patients had a concomitant kidney biopsy performed at the time of urine collection. In addition, patients with other renal diatheses were also included as controls.All three molecules were elevated in the urine of systemic lupus erythematosus patients, although VCAM-1 (area under curve = 0.92) and MCP-1 (area under curve = 0.87) were best at distinguishing the systemic lupus erythematosus samples from the healthy controls, and were also most strongly associated with clinical disease severity and active renal disease. For patients in whom concurrent renal biopsies had also been performed, urine VCAM-1 exhibited the strongest association with the renal pathology activity index and glomerulonephritis class IV, although it correlated negatively with the chronicity index. Interestingly, urinary VCAM-1 was also elevated in anti-neutrophil cytoplasmic antibodies-associated glomerulonephritis, focal segmental glomerulosclerosis and membranous nephropathy but not in minimal-change disease.Urinary VCAM-1 emerges as a reliable indicator of the activity:chronicity ratios that mark the underlying renal pathology in lupus nephritis. Since VCAM-1 is involved in the acute phase of inflammation when leukocytic infiltration is ongoing, longitudinal studies are warranted to establish whether tracking urine VCAM-1 levels may help monitor clinical and pathological disease activity over time. Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease with the potential to affect a variety of end organs. Lupus nephritis (LN) is one of the most frequent manifestations of SLE and can be present in 60% of SLE patients . LN is aSeveral studies of murine models of LN as well as studies in SLE patients have uncovered a number of potential disease biomarkers - including chemokines, cytokines and adhesion molecules - that may correlate well with LN -9. HowevMonocyte chemotactic protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1) and CXC ligand 16 (CXCL16) have been documented to be increased within the kidneys, sera and urine of SLE patients and seem to correlate with disease activity, although their clinical utility in predicting disease activity in LN remains to be fully established ,9,12-15.VCAM-1 is an adhesion molecule involved in the migration and recruitment of inflammatory cells through its interaction with an integrin (very late antigen-4) present on infiltrating leukocytes. In murine lupus, VCAM-1 has been shown to be hyperexpressed in the endothelium, in the glomeruli and in the tubules of MRL.lpr mice , as wellUrinary MCP-1, VCAM-1 and CXCL16 thus emerge as promising markers of LN, with independent confirmation of these findings by two or more groups. However, few studies have been designed to assess urinary biomarker levels in relation to the status of concurrent renal pathology in LN. To address this lack of data, 74 SLE patients with biopsy-proven LN seen at UT Southwestern Medical Center, Dallas, TX, USA were studied for urinary levels of MCP-1, CXCL16 and VCAM-1. Of these, 24 patients had urine samples drawn at the same time as the renal biopsy was performed. This offered us the unique opportunity to identify the markers that were most predictive of the class of glomerulopathy as well as renal pathology activity/chronicity indices in LN. In addition, we also included as controls patients with anti-neutrophil cytoplasmic antibodies-associated glomerulonephritis (ANCA-GN), focal segmental glomerulosclerosis (FSGS), membranous nephropathy and minimal-change disease, in order to assess the specificity of any observed associations with LN.The study was approved by the UT Southwestern Institutional Review Board. Patients with LN who were followed up at the outpatient clinic or were hospitalized at UT Southwestern Medical Center were consented and enrolled in the study if they had biopsy-proven LN during the course of their follow-up. Included were subjects who underwent kidney biopsy during the study enrollment period, before they were started on immunosuppressive treatment for their LN.SLE disease activity at the time of sample procurement was calculated using the SELENA Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) . Renal SELISA kits for assaying MCP-1, CXCL16 or VCAM-1 were purchased from R&D Laboratories  or Cayman Chemicals  and were used as indicated by the manufacturer . All urine samples were diluted 1:1 or more for the ELISA, and the concentrations of the respective molecules were ascertained from standard curves constructed using manufacturer-supplied standards. The urine protein:creatinine ratio was used as an estimate of 24-hour proteinuria, and the urinary levels of all three molecules assayed were normalized against the corresponding urine creatinine levels.Renal pathology was reported using the standardized International Society of Nephrology/Renal Pathological Society classification . In addit test where the data were normally distributed. Otherwise, the nonparametric Mann-Whitney U test was used. Statistical tests including correlation coefficients (R) were calculated using Graph Pad Prism. P < 0.05 was considered significant.Groups were compared against each other using the Student's There were 74 LN patients, 13 healthy volunteers and 22 disease controls enrolled in this study. One patient was excluded since he had progressed to end-stage renal disease and was on dialysis at the time of sample procurement. There were 12 males and 61 females. Thirty-three participants were African Americans and 33 participants were Hispanics, with the remaining seven being whites. All patient characteristics are detailed in Table R = 0.82, P < 0.0001). Although both urinary MCP-1 (mean 542.9 ± 183.5 pg/mg) and CXCL16 (mean 24.8 ± 6.19 pg/mg) were significantly correlated with proteinuria, the correlation was smaller compared with that for urinary VCAM-1 . Although all three molecules were elevated in the urine of SLE patients, they showed interesting differences particularly when the patients were segregated based on ethnicity . Among the nonactive nephritis patients, therefore, VCAM-1, CXCL16 and MCP-1 levels and the urine protein:creatinine ratio were not different between the patients who received a specific medication and those who did not. Likewise, among active nephritis patients, VCAM-1 and CXCL16 levels as well as the urine protein:creatinine ratio were not different between the patients who received a specific medication and those who did not.To evaluate whether concurrent medications might impact the level of these urinary markers, we first examined whether the medications used were different between the patients with active nephritis and the patients without. As shown in Table A second phase of analysis was carried out, focusing on a unique subset of the recruited LN patients. Since the urine samples for 24 of the participating subjects were obtained at the time of kidney biopsy, we correlated the renal pathology with the corresponding levels of the assayed markers. When the urinary levels of the three molecules in patients with class IV LN were compared against the corresponding levels in patients with other classes of LN, all three markers exhibited higher levels in class IV LN and tended to differentiate this class from other classes of LN Figure . SimilarFinally, we wanted to assess the disease specificity of urinary VCAM-1. Although our data clearly indicate that patients with active LN had the highest levels of urinary VCAM-1, whether other types of inflammatory or non-inflammatory nephropathies may also be associated with elevated VCAM-1 levels is unclear. Interestingly, the urinary levels of VCAM-1 in patients with ANCA-GN, FSGS and membranous nephropathy were not statistically different from the urine levels of VCAM-1 in patients with active LN Figure . In contThe management of LN remains an intricate problem ,27 sinceWhen the urinary levels of the three molecules were analyzed in patients who had a concurrent kidney biopsy performed, the levels of all three molecules were elevated in class IV LN and tended to differentiate class IV patients from the subjects with other classes of LN, again with VCAM-1 being the most discriminatory. This is an important finding since class IV LN has ominous prognosis, warranting prompt and aggressive treatment. An interesting finding emerged when we examined the relationship of these markers against the corresponding renal pathology AI in concurrently performed renal biopsies. All three molecules correlated positively with the AI, but only the correlation for urine VCAM-1 was statistically significant. Since the AI signifies severe but potentially reversible inflammation, this is an important finding with respect to disease monitoring and treatment. Similarly, all three markers showed a trend towards a negative correlation with CI - high chronicity indices denote late-stage fibrosis, minimal inflammation and irreversible damage to the kidney, all of which bode poorly even with treatment.Taken together, these findings suggest that urinary VCAM-1 may be a reliable indicator of the activity:chronicity ratios that mark the underlying renal pathology in LN. In comparison with VCAM-1, urine MCP-1 performed modestly in its predictive capacity while CXCL16 was even less promising as a disease marker. VCAM-1 is an important adhesion molecule involved in the key process of leukocytic migration into the kidneys. Leukocytic infiltration is a hallmark of severe renal disease and one of the morphological features contributing to the elevated AI in LN. Since LN is involved in the acute phase of inflammation when leukocytic infiltration is ongoing and since VCAM-1 levels are likely to recede with reduced activity and when chronicity sets in, tracking VCAM-1 levels longitudinally may help monitor disease activity over time.However, our findings indicate that elevated urinary VCAM-1 is not specific for SLE; rather, it appears to be a marker of renal injury, since the levels are also elevated in other types of inflammatory nephritis  as well as nephropathies not typically associated with inflammation . Although the numbers of each type of renal disease control are rather limited, and are not completely matched with the LN patients with respect to renal function, age or gender, these preliminary findings warrant a more thorough investigation of urinary VCAM-1 levels in other renal diatheses. In addition, the clinical utility of using urine VCAM-1 to track disease progression in ANCA-GN, FSGS and membranous nephropathy should also be carefully examined in future studies. To this end, the increased expression of VCAM-1 either within the kidneys or in the urine has already been documented previously in patients with ANCA-GN, FSGS and membranous nephropathy, in resonance with our findings -36. InteThe clinical use of urinary VCAM-1 clearly must be coupled with a second marker that can accurately predict chronicity changes within the kidney. In this light, although markers such as transforming growth factor beta offer promise , severalThree urinary molecules and concurrent renal pathology were examined in SLE. Urinary VCAM-1 emerges as a good indicator of the renal pathology AI in patients with LN.AI: activity index; ANCA-GN: anti-neutrophil cytoplasmic antibodies-associated glomerulonephritis; CI: chronicity index; CXCL16: CXC ligand 16; ELISA: enzyme-linked immunosorbent assay; FSGS: focal segmental glomerulosclerosis; LN: lupus nephritis; MCP-1: monocyte chemotactic protein-1; SLE: systemic lupus erythematosus; SLEDAI: Systemic Lupus Erythematosus Disease Activity Index; VCAM-1: vascular cell adhesion molecule-1.This work was supported in part by the George M. O'Brien Kidney Research Core Center  and by the Lupus Research Institute. The authors declare that they have no competing interests.SS, TW, CX, KV and JH executed the studies. SS, TW, TM, HBO and CA contributed to data analysis and writing the manuscript. XJZ performed the pathological analysis. CP and RS contributed patient material and data. CP, RS and CM contributed to planning the study and preparing the manuscript. All authors read and approved the final manuscript.


Five persons in France were infected with Orf virus after skin wounds were exposed to infected sheep tissues during Eid al-Adha, the Muslim Feast of Sacrifice. Infections were confirmed by electron microscopy, PCR, and sequence analysis. Prevention and control of this underdiagnosed disease can be achieved by educating physicians, slaughterhouse workers, and persons participating in Eid al-Adha. Parapoxvirus, family Poxviridae) is endemic to most countries. The virus primarily causes contagious ecthyma in wild and domestic ruminants, mostly sheep and goats  for an 8-mm lesion without local complications on her left thumb. Case-patient 2, the 33-year-old niece of case-patient 1, was hospitalized on November 29 for surgical excision of a phlegmonous lesion on the fifth finger of her left hand; she also had a fever and a lesion without local complications on the second finger of the same hand. The surgery was performed by CF, the same surgeon who examined case-patient 1. By chance, case-patient 3, the 38-year-old brother of case-patient 2, met CF on November 29 while visiting his sister in the hospital; he asked CF if she would examine lesions on his left hand. Clinical examination revealed 2 lesions (1 each on the thumb and third finger) without local complications , the Muslim Feast of Sacrifice. Using a smartphone, CF photographed the lesions on case-patient 3 and transmitted the photographs to 2 infectious disease specialists. The specialists indicated that the lesions appeared to be typical of parapoxvirus infection. CF interviewed case-patients 1–3 again, and they reported having knife wounds after preparing lambs for the religious feast and seeing lesions on the gums and tongue of 1 lamb. Swab  specimens were obtained from lesions on case-patients 1 and 3 and sent, along with a surgical skin biopsy specimen from case-patient 2, to the virology laboratory at Public Assistance–Hospitals of Marseille, Marseille, France.Case-patient 4, a 64-year-old woman, sought medical care on December 9 for an ulcerovegetative lesion on the third finger of her left hand; the lesion was on the internal face of the interphalangeal joint, and phlegmon and cellulitis were present. The patient reported that she had injured herself with a kitchen knife on November 6 while butchering lamb meat for Eid al-Adha. Ten days later, she noticed vesicular lesions at the injury site; a pustule complicated by superinfection subsequently developed. The lesion was surgically excised on December 12, and skin biopsy samples were sent to the virology laboratory at Public Assistance–Hospitals of Marseille.Case-patient 5, a 42-year-old woman, sought medical care on December 14 for a painful 2-cm papulonodular lesion on her right wrist , panel CSpecimens from case-patients 1–3 were received at the laboratory on December 2. Negative-stain electron microscopy (EM) was immediately performed: images revealed typical poxvirus-like particles in the specimen from case-patient 3 , panel D2 flasks; none of the samples yielded infectious virus. We later attempted virus isolation again, using fetal bovine esophagus cells, and isolated Orf viruses from samples from case-patients 3 and 5.Immediately after Orf virus infection was confirmed, we attempted virus isolation by cell culture, using Vero cells (the only cells available at the time) in 12.5-cmThe 4 partial sequences obtained from samples from case-patients 1 and 3–5 were identical (GenBank accession no. JQ596637). We used ClustalX .County veterinary services traced the origins of the sheep considered to be responsible for these human cases of Orf virus infection. The first 3 cases were linked to an illegal slaughterhouse within the county where sheep from France and Spain had been housed for 1 month. Case 4 was linked to sheep carcasses that were purchased from 2 legally operating butchers. Case 5 was linked to a certified temporary slaughterhouse that had sheep from regional counties, Spain, and Romania.Although human Orf virus infections have typically been associated with occupational animal contact  were initially reserved for the ritual sacrifice during Eid al-Adha. Today, however, Muslims in developed countries  mostly buy lambs, which are cheaper and more plentiful but also highly susceptible to Orf virus infections (The cases reported here stress the need for using appropriate measures to prevent animal-to-human transmission of pathogens. Public health officials should educate persons with occupational or household exposure to animals about the possibility for disease transmission and ways to avoid infection. Persons at increased risk for exposure to Orf virus include livestock owners, slaughterhouse workers, and persons who prepare animals at home for religious practices. Persons who handle animals should wear nonpermeable gloves, avoid exposure of open wounds, and meticulously wash skin wounds with soap and water after handling animals (Phylogeny of Orf virus sequence of virus isolates and results of agarose gel electrophoresis of PCR products from virus isolates from Orf virus–infected persons, Marseille, France.


Dendritic cells (DCs) are antigen presenting cells capable of inducing specific immune responses against microbial infections, transplant antigens, or tumors. Interestingly, microenvironment conditions such as those present in tumor settings might induce a DC phenotype that is poorly immunogenic and with the capability of promoting angiogenesis. We hypothesize that this plasticity may be caused not only by the action of specific cytokines or growth factors but also by the properties of the surfaces with which they interact, such as extracellular matrix (ECM) components.Herewith we studied the effect of different surfaces and soluble factors on the biology of DCs. To accomplish this, we cultured murine myeloid(m) DCs on surfaces coated with fibronectin, collagen I, gelatin, and Matrigel using poly-D-lysine and polystyrene as non-biological surfaces. Further, we cultured these cells in the presence of regular DC medium (RPMI 10% FBS) or commercially available endothelial medium (EGM-2). We determined that mDCs could be kept in culture up to 3 weeks in these conditions, but only in the presence of GM-CSF. We were able to determine that long-term DC cultures produce an array of angiogenic factors, and that some of these cultures still retain the capability to induce T cell responses.Altogether these data indicate that in order to design DC-based vaccines or treatments focused on changing the phenotype of DCs associated with diseases such as cancer or atherosclerosis, it becomes necessary to fully investigate the microenvironment in which these cells are present or will be delivered. Interestingly, DCs have been shown to possess a remarkable cellular plasticity. For example, pDCs could acquire myeloid DC characteristics under the influence of viral infection  (ABTS) substrate system . The blue-green color produced by enzymatic activity was quantified at 405 nm in an ELISA microplate reader .5/well in RPMI containing 10% FBS. Spleens were resected from healthy BALB/c mice and minced in a sterile fashion to yield a single cell suspension and erythrocytes were eliminated by hypotonic shock. Then untouched CD3 T cells were purified from this suspension by magnetic sorting using the Pan T Cell Isolation Kit (MACS Miltenyi) following the manufacturer's instructions. T cells were labeled with CFSE as previously described [5 cells/well with the recovered DCs for 5 days. CFSE dilution, an indication of cell proliferation, was assessed by qualitative flow cytometry analysis of gated CD3 T cells.Murine myeloid C57BL/6 DCs recovered from long-term cultures and treated for 48 h with an inflammatory cocktail as described above were seeded in 96-well round-bottom plates at a concentration of 1 × 10escribed  and wereFor multiple comparisons we performed ANOVA analysis with post-analysis by the Tukey-Kramer multiple comparisons test or the Dunnett's post comparison test. A value of p < 0.05 was considered significant. Data are expressed as mean ± SD. Data was analyzed by using the Graph Pad Instat software .In the studies reported herewith, we used murine bone marrow-derived (myeloid) DCs. Figure in vivo under pathological conditions [Murine myeloid(m) DCs have been extensively used in order to determine the efficacy and improvement of DC-based vaccines; investigate DC:T cell interactions or DC development; and determine their role in pathological conditions such cancer or infectious diseases -36. Thusnditions . We hyponditions . As showAltogether these data indicate that similar to what has been reported in the human -40, muriWe have previously shown that murine mDCs could be cultured on Matrigel in the presence of tumor factors for long periods of time . SimilarUpon one week of culture in different conditions we performed a qualitative photographic analysis of the attached mDCs. As shown in Figure In order to further determine the effect of different culture conditions on the biology of mDCs, we evaluated the expression of costimulatory molecules by qualitative flow cytometry analysis on CD11c gated cells. As depicted in Figure In a series of complementary studies, we investigated the supernatants of these cells for inflammatory cytokines and nitrites. Upon treatment with LPS and TNFα, mDCs are able to generate inflammatory molecules such as IL-6 and IL-1α, and to upregulate the expression of nitric oxide synthase-II, which generates nitric oxide. As shown in Figure Taking into account previous data showing that DCs associated with pathological conditions are able to produce angiogenic factors ,41 we deFinally, DCs cultured for one week on different surfaces and media conditions express CD54 as determined by comparison with isotype controls in a qualitative flow cytometry assay Figure . This moin vitro through the method described by Lutz [In our previous experiments both RPMI and EGM cultures were supplemented with external GM-CSF. Myeloid DCs obtained  by Lutz  are diff by Lutz , when mD by Lutz . As depi by Lutz . On the in vivo. To accomplish this, mouse ovarian tumors were developed in C57BL/6 mice, dissected and RNA was extracted from whole tumor samples and analyzed by real-time quantitative PCR. As determined by ANOVA analysis followed by Dunnett post-comparison tests, we were able to determine that all tumor samples except one expressed GM-CSF at levels that were not significantly different from those observed in normal murine spleen, an immunological organ used as a control in these studies  , hepatocIn a complementary series of studies, we analyzed by qualitative PCR the presence of additional angiogenic factors and receptors in these cells. As shown in Figure Herewith we decided to focus our studies on the immune properties of mDCs cultured for 3 weeks with EGM on different surfaces. The rationale being that in previous studies endothelial medium seemed to skew the cells farther away from a typical immunostimulatory DC phenotype ,57. As sTo further determine the immune capabilities of these cells, after stimulation with an inflammatory cocktail we cocultured them with CFSE-stained allogeneic BALB/c T lymphocytes. Consistently with the ELISA and FACS data, CD11c cells recovered from Matrigel and poly-D-Lysine cultures induce higher levels of proliferation of allogeneic T cells than cells recovered from other conditions Figure . Indeed,DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of MHC molecules. Typically, antigen-loaded DCs migrate to immunological organs where they present the processed antigens to T lymphocytes thus triggering specific immune responses. On the other hand, a role of DCs as promoters of angiogenesis under pathological conditions has been suggested ,15,58,59in vitro characterized by the loss of CD14/CD45 and upregulation of endothelial markers such as CD31, CD34, von Willebrand factor, VEGF receptor (VEGFR)-2 and VE-Cadherin [bonafide endothelium such as LDL uptake, lectin binding or formation of cord-like structures in 3D gels [We and others have shown that monocytes or DCs can undergo an endothelization process Cadherin ,41,60. T 3D gels ,60,61. D 3D gels ,58,62-64Several reports indicate that soluble factors can alter the biology of DCs. Tumor-associated cytokines such as VEGF, IL-10 and PGE-2 can profoundly affect the nature of APCs. In particular, DCs showing low levels of costimulatory molecules have been detected in tumors expressing high levels of VEGF  and tumoet al. [et al. [et al., [On the other hand, few studies have been undertaken in order to determine the effect of different ECM components on the biology of DCs. Foundational work on this subject was performed on 1998 by Brand et al. . This re [et al.  describe[et al.,  reportedHerewith we report that attachment to a surface together with factors provided in the media allowed long-term mDC cultures to exhibit different immunological capabilities. DCs are usually considered short-lived, terminal cells whose main function is to activate T cells in order to induce specific immune responses. We were able to demonstrate that these cells need the presence of GM-CSF in order to be able to establish long-term cultures; that GM-CSF can be produced by ovarian cancer cells and that is expressed in the microenvironment of mouse ovarian tumors. In terms of the role of adhesion surfaces on the biology of these cells, we were able to show that cells cultured on different surfaces showed expression of angiogenic molecules such as HGF, bFGF, MMP2 and MMP9. We also showed that these cells are able to produce VEGF-A, -B and PIGF. Taking into account that most of these molecules were expressed in every condition it is tempting to speculate that if mDCs are not driven to initiate an immune response they will by default remain in a proangiogenic state. Further, since we were able to detect the expression of VEGFR-1 and -2 in these cells it is tempting to speculate that some of these angiogenic molecules might act in an autocrine fashion, promoting cell survival or induction of other angiogenic molecules. Finally, expression of MMPs by DCs might promote VEGF availability  thus conIn terms of immunological properties, we observed that after 1 week culture in non-inflammatory conditions, mDCs showed low levels of costimulatory molecules. Attachment to the different surfaces did not inhibit the capability of these cells to respond to inflammatory stimuli. In particular, these cells upregulated the expression of costimulatory molecules, and produced IL-1α, IL-6 and nitric oxide when cultured in the presence of LPS and TNFα. This effect was less pronounced when cells were cultured with EGM, indicating that the factors present in this medium can decrease the effects of inflammatory factors. Further, DCs cultured on different surfaces for 3 weeks showed differential immunological capabilities. In particular, DCs recovered from Matrigel, (a gelatinous protein mixture purified from murine tumor stroma) and from poly-D-Lysine  were able to respond to maturation stimuli similarly to fresh mDCs. Upon stimulation, these cells produced large quantities of IL-1α and IL-6, and induced proliferation of allogeneic lymphocytes. On the other end, DCs recovered from collagen I surfaces were completely unresponsive to maturation stimuli.Herewith we demonstrated that the combination of soluble factors together with adhesion surfaces determines particular mDC profiles. Thus, in order to design DC-based vaccines or treatments focused on changing the phenotype of DCs associated with diseases, such as cancer -70 or atin vivo studies, FACS analysis, direction of coworkers, experimental design and writing. All authors read and approved the manuscript.LS: ELISA analysis, qualitative and quantitative PCR; MM: qualitative and quantitative PCR: analysis, immunohistochemistry analysis; MP: ELISA analysis, qualitative and quantitative PCR, direction of coworkers; EM: qualitative PCR analysis; J.M: qualitative PCR analysis; HN: Immunohistochemistry, quantitative PCR; AKV: ELISA analysis; AR: ELISA Analysis MG and OO: Nitrite analysis; KM: qualitative PCR analysis; MCC: FACS analysis; FB: Cell culture, sample collection,


Patients with narcolepsy-cataplexy (NC) mostly exhibit undetectable hypocretin levels. Hypocretin system is one of the key players in the complex interaction between sleep and the cardiovascular system. We tested the hypothesis that hypocretin deficiency affects cardiovascular risk factors by measuring nighttime and daytime ambulatory blood pressure (BP) and the night-to-day BP ratio as well as endothelial dysfunction by the digital pulse amplitude response in drug-free patients with NC compared to controls.Sleep, clinical and biological cardiovascular risk factors, fingertip peripheral arterial tonometry, and 24-hour ambulatory BP monitoring were recorded in 50 drug-free patients with NC and 42 healthy control subjects, except for BP monitoring available in all controls but in 36 patients with NC.More patients than controls were smokers, obese and with dyslipidemia. One-third of patients with NC were “non-dippers” (defined as <10% drop in BP during sleep) compared to only 3% of controls. The diastolic non-dipper BP profile had up to 12-fold higher odds of being associated with NC. We noted negative correlations between mean diastolic BP fall during night, REM sleep percentage and number of sleep onset REM periods, and a positive correlation with mean sleep latency on the MSLT. The digital pulse amplitude response measured by fingertip was similar between NC and controls.We found a high percentage of non-dippers in patients with NC with association with REM sleep dysregulation. The blunted sleep-related BP dip in NC may be of clinical relevance, as it may indicate increased risk for cardiovascular events. Narcolepsy with cataplexy (NC) is characterized by excessive daytime sleepiness (EDS), cataplexy, and disturbed nocturnal sleep, including parasomnias, obstructive sleep apnea syndrome, and periodic leg movements Marked decreases in hypocretin-1 in the cerebrospinal fluid and in the number of hypocretin neurons have been demonstrated in NC Animal studies have explored the cardiovascular status of hypocretin-deficient rodent during wakefulness, showing lower arterial blood pressure (BP) compared to wild type Despite frequent associations between NC and obesity, type 2 diabetes mellitus, and metabolic syndrome, few studies addressing cardiovascular consequences have been conducted in human NC Because hypocretin system is one of the factors involved in the complex interaction between sleep and the cardiovascular system, we tested the hypothesis that hypocretin deficiency may affect markers of cardiovascular risk. We measured nighttime and daytime ambulatory blood pressure (BP) and their night-to-day ratio, the digital pulse amplitude response (RH-PAT) to evaluate endothelial dysfunction in drug-free patients with narcolepsy-cataplexy (NC) compared to controls.All patients and controls gave their informed written consent to take part in the study which was approved by the Local Ethics Committee . Additional written consent was obtained from the parents of participants under the age of 18.Fifty patients with typical NC  were examined. NC was diagnosed according to the revised International Classification of Sleep Disorders Forty-two community-dwelling healthy control participants  were recruited from local associative networks .No patients or controls had any psychiatric disorder, based on DSM-IV TR criteria, and none was taking any medication known to influence sleep at the time of the study. Regarding the presence of medication influencing cardiovascular function, five subjects (four patients with NC and one control) were treated with an antihypertensive drug, two patients with a drug for diabetes, and one patient with a treatment for hyperlipidemia.All participants underwent a routine medical history and physical examination, and body-mass index (BMI) and waist and hip circumference were measured. All participants fasted for venous blood sampling between 7 and 8 AM, with similar procedures to assess for cardiovascular biological risk factors.Presence of baseline cardiovascular risk factors was defined as follows: (1) hypertension as a conventional BP of at least 130 mm Hg systolic and 85 mm Hg diastolic, or as use of antihypertensive drugs; (2) diabetes mellitus as positive patient history, a fasting blood glucose concentration of at least 7.0 mmol/L, or use of antidiabetic drugs; (3) hyperlipidaemia as total serum cholesterol >240 mg/dL, triglyceride ≥150 mg/L, or treatment with lipid-lowering drugs; (4) metabolic syndrome diagnosed according to National Cholesterol Education Program Adult Treatment Panel III criteria All patients (n = 50) and 23 controls underwent one night of polysomnography (PSG) recording in the sleep laboratory followed by the Multiple Sleep Latency Test (MSLT) the next day. Sleep recording included 3 EEG leads , 2 electroculograms, one chin electromyogram, and an electrocardiogram. Airflow was measured using a nasal cannula and mouth thermocouple, thoracoabdominal movement by respiratory belts (piezoelectric), and arterial oxygen saturation by finger pulse oximetry. Sleep recordings were manually scored by standard criteria for sleep stages as well as microarousals and respiratory events Digital pulse amplitude was measured between 7 and 8 AM after overnight fasting in all participants in supine position by RH-PAT using an Endo-PAT finger sensor  Twenty-four-hour ambulatory blood pressure monitoring (ABPM) was done on the non-dominant arm using a validated automatic oscillometer  following the PSG-MSLT recording in 36 patients and in all controls (n = 42). All participants were instructed to continue their usual daily activities but without napping. BP and HR readings were automatically recorded at 15-minute intervals in daytime and 60-minute intervals at night. Daytime and nighttime were individually predetermined depending on the participants’ usual awake and sleep schedule. Recordings were analyzed to obtain average 24-hour daytime and nighttime systolic and diastolic BP and HR. Systolic and diastolic BP were used to assess dipping. A “non-dipping BP profile” was defined as a nocturnal BP dip<10% lower than daytime BP Categorial variables for the sample are presented as percentages, and quantitative variables as medians with ranges. Distributions were mostly skewed, according to Shapiro-Wilk’s test. Chi square or Fisher’s exact tests were run to compare categorical variables between the two groups, and the Mann-Whitney test for continuous variables . SpearmaAs expected, Regardless of the distribution , RH-PAT 90–120 measurements were not significantly lower in the NC group than in controls, before and after adjusting for gender, smoking, and BMI  . RH-PAT Patients with NC showed lower daytime diastolic BP and HR than controls, with similar tendency for the mean daytime pressure . Large bWe noted in the whole population a negative correlation between mean diastolic dip and REM sleep percentage  and number of SOREMPs , and a positive correlation with mean sleep latency on the MSLT . No association was found between RH-PAT, and mean 24-h blood pressure or dipper status.For the first time, 24-hour ambulatory blood pressure (BP) monitoring and the peripheral endothelial function were reported in a large group of drug-free NC patients in comparison to controls. Although the digital pulse amplitude response, measured by reactive hyperemia-peripheral artery tonometry (RH-PAT), was substantially similar between NC and controls, one-third of NC patients were non-dippers vs. only 3% of controls. Non-dipper diastolic BP status was strongly associated with NC by an up to 12-fold higher odds ratio, with significant associations with nocturnal and diurnal REM sleep, and objective daytime sleepiness.Patients with NC showed lower daytime diastolic BP, and HR than controls. This could be partially explained by lesser physical activity in patients due to severe daytime hypersomnia We recently reported lower cardiac activation associated with periodic leg movements during sleep (PLMS) in NC, probably due to changes in baroreflex sensitivity One strength of this study is the between-group comparison of robust cardiovascular markers in drug-free patients with NC by considering during meaning time potential clinical and biological confounding factors. Patients with NC were more smokers, obese and with dyslipidemia than controls but without any difference for hypertension, diabetes mellitus, metabolic syndrome, insulin resistance, and personal history of coronary artery disease or stroke. We found associations between diastolic non-dipper status and dyslipidemia in NC with a similar tendency for REM sleep percentage A reliable, non-invasive method using a PAT sensor was developed to detect vascular changes in patients at high cardiovascular risk Unlike a recent study In conclusion, we demonstrated a large percentage of diastolic non-dippers in patients with NC compared to controls, with associations with REM sleep regulation. The blunted sleep-related BP dip in NC may be of clinical relevance as it may indicate increased risk for cardiovascular events.


Understanding the factors that impact on disability is necessary to inform trauma care and enable adequate risk adjustment for benchmarking and monitoring. A key consideration is how to adjust for pre-existing conditions when assessing injury outcomes, and whether the inclusion of comorbidity is needed in addition to adjustment for age. This study compared different approaches to modelling the impact of comorbidity, collected as part of the routine hospital episode data, on disability outcomes following orthopaedic injury.12-month Glasgow Outcome Scale – Extended (GOS-E) outcomes for 13,519 survivors to discharge were drawn from the Victorian Orthopaedic Trauma Outcomes Registry, a prospective cohort study of admitted orthopaedic injury patients. ICD-10-AM comorbidity codes were mapped to four comorbidity indices. Cases with a GOS-E score of 7–8 were considered “recovered”. A split dataset approach was used with cases randomly assigned to development or test datasets. Logistic regression models were fitted with “recovery” as the outcome and the performance of the models based on each comorbidity index (adjusted for injury and age) measured using calibration (Hosmer-Lemshow (H-L) statistics and calibration curves) and discrimination (Area under the Receiver Operating Characteristic (AUC)) statistics.All comorbidity indices improved model fit over models with age and injuries sustained alone. None of the models demonstrated acceptable model calibration . There was little difference between the discrimination of the indices for predicting recovery: Charlson Comorbidity Index ; number of ICD-10 chapters represented ; number of six frequent chronic conditions represented ; and the Functional Comorbidity Index .The presence of ICD-10 recorded comorbid conditions is an important predictor of long term functional outcome following orthopaedic injury and adjustment for comorbidity is indicated when assessing risk-adjusted functional outcomes over time or across jurisdictions. Measuring disability following injury is important. Understanding the factors that impact on disability is necessary to inform trauma care, and enable adequate risk-adjustment for benchmarking and monitoring. The presence of comorbidities has been identified as an important predictor of mortality following injury -3. Otheret al., identified numerous methods for measuring comorbidity, and highlighted many issues with the development and validation of comorbidity indices [De Groot  indices . Of note indices . Several indices ,10-16 buThis study compared different approaches to modelling the impact of comorbidity, collected as part of the routine hospital discharge data, on 12-month disability outcomes in an orthopaedic injury population to inform the development of risk adjustment models for disability outcomes. A key additional aim was to establish whether the inclusion of comorbidity provides additional important predictive value in addition to adjustment for age alone.The Victorian Orthopaedic Trauma Outcomes Registry (VOTOR) monitors the management and outcomes following admission to hospital for adults with acute orthopaedic injury . This seCases included survivors to hospital discharge, admitted between March 2007 and July 2010. Demographic, injury event, International Classification of Diseases 10th revision Australian Modification (ICD-10-AM) diagnosis codes (up to 40 per admission), and 12-month functional outcomes data were extracted for analysis.ICD-10-AM orthopaedic injury diagnosis codes were mapped to 10 orthopaedic injury groups. Indicator variables were generated to represent important non-orthopaedic injuries, including variables for intracranial injury and/or skull fracture, multiple rib fractures, intra-abdominal or intra-thoracic organ injury, and burns.ICD-10-AM diagnosis codes are the source of comorbidities for VOTOR. In Victoria, each code contains a prefix with “P” representing principal diagnosis requiring treatment during the stay, “A” representing additional diagnoses and “C” representing in-hospital complications. Australian Coding Standards specify that most conditions should be coded as additional diagnoses if they affect treatment, require investigation, or use resources during the admission. However, the standards require coding of certain conditions whenever they are present; some communicable diseases , diabetes and pregnancy .For the purposes of this study, the following were excluded from mapping to comorbidity measures:i. All in-hospital complications (“C” prefix codes);ii. Chapter XIX  with a “P” prefix ;iii. All Chapter XVIII  as these are not comorbidities;iv. Chapters XX  to XXII  as these are not comorbidities.Remaining “P” and “A” prefixed codes were checked by the authors  to remove erroneously coded complications. These included acute post-haemmorhagic anaemia, acute subendocardial myocardial infarction, acute renal failure, acute respiratory infections, fever and post-traumatic amnesia. Codes remaining were assumed to represent comorbidities.et al.[Four methods for classifying comorbidity were used and the ICD-10-AM codes mapped to the four indices. These were: (i) Charlson Comorbidity Index (CCI); (ii) Functional Comorbidity Index (FCI); (iii) categorisation by ICD-10 chapter; and (iv) the six conditions described by Haagsma et al..The 19 Charlson conditions were mapped to the CCI from the ICD-10-AM codes  self-administered questionnaire where the FCI score is the sum of the number of conditions reported for the person (0–18) . ICD-10-et al.[Indicator variables were generated for the presence or absence of conditions in Chapters I to XVII, and Chapter XIX. A variable indicating the number of chapters represented was generated, an approach used by Cameron et al..et al. investigated the impact of comorbidity on disability weight estimates in a sample of 2,295 injured patients in the Netherlands [Haagsma herlands . The sixThe key differences between the comorbidity indices used relate to the number of conditions represented. The ICD chapter approach maps all available ICD-10 comorbidity codes into 18 chapter-related groups, but does not specifically identify individual conditions. For example, diabetes is included in the “Endocrine, nutritional and metabolic disorders” chapter. The six frequent chronic conditions described by Haagsma et al.  also useall conditions specified by the six frequent chronic condition, FCI and CCI comorbidity measures. All remaining measures include specific categories for heart disease, chronic pulmonary disease, and diabetes, although how this is represented varies  statistic and calibration curves. A higher H-L statistic and significant p-value correspond to poorer calibration . CalibraThe area under the receiver operating characteristic (ROC) curve (AUC) measures the capacity of the model to discriminate between those who do and do not experience the outcome of interest . ROC ploIn the training dataset, a model was fitted with injury variables and age as independent variables, and “recovery” as the dependent variable. Each measure of comorbidity was added and the models were compared using likelihood ratio (LR) tests, AUC (95% CI), and the H-L statistic. The following comorbidity adjustment methods were used.i. CCI weight categorised as 0, 1, ≥ 2ii. Number of ICD-10 chapters represented categorised as 0, 1, ≥ 2iii. FCI score categorised as 0, 1, ≥ 2iv. Number of the six frequent chronic conditions described by Haagsma et al.  represenv. All indicator variables for ICD-10 chapters includedvi. All FCI condition indicator variables includedvii. All of the six frequent chronic condition indicator variables included.The number of conditions/weighted index was not linearly related to the log odds of recovery, requiring categorisation. The individual conditions of the CCI were not modeled as the weighted index is the most commonly used form of the index. Models were then fitted to the test dataset and fit assessed using the AUC, H-L statistics, and calibration curves. Data were complete for all data items included in the models, ensuring that comparison between models was based on the same cases. All analyses were performed using Stata Version 11.0 . A p-value <0.05 was considered significant.There were 15,471 survivors to discharge, and 13,519 (87.4%) had a valid GOSE-E at 12-months. Cases lost to follow-up at 12-months included a higher proportion of patients less than 45 years of age, male, and injured in motor vehicle crashes , FCI  or the six frequent chronic conditions . The AUC was higher for the number of ICD-10 chapters represented when compared to the number of the six frequent chronic conditions represented . However, the overall range of the AUC for the models adjusting for comorbidity ranged from 0.716 to 0.729 , confirming the importance of other factors. Numerous injury, personal and environmental factors have the capacity to influence the recovery of an individual. This is well demonstrated in the literature where factors such as compensable status ,16,38,39The strengths of this study are the large number of cases , high follow-up rate at 12-months , and the use of a hospital discharge data to identify comorbidities which has been shown to have low error rates in diagnoses audited . Howeveret al.) to ICD-10 based datasets. In future, the development of privacy protecting record linkage systems may enable primary care data to be linked to trauma and hospital discharge datasets to permit evaluation of the impact of conditions not deemed to influence in-hospital care.The study focused on orthopaedic trauma cases rather than all admitted trauma cases. While many cases had also sustained non-orthopaedic injuries, the results may not reflect cases without orthopaedic injury and this should be considered when interpreting the findings. Nevertheless, the orthopaedic registry was selected as the data source specifically for this study because this population includes a higher proportion of elderly patients with comorbidities than other injury populations such as major trauma. There were differences between included cases and those lost to follow-up at 12-months with a bias towards older patients with comorbidity in the group followed-up and included in this study. Secondly, there are challenges in collecting comorbidity information from patients from large populations and particularly where cognitive deficit (e.g. head injury and pre-existing dementia) are prevalent. Therefore, we were not able to assess the agreement between self-report and ICD-10 coding, or fully evaluate the relationship between the FCI and the six frequent chronic conditions not prevalent in the ICD-10 codes, highlighting issues with adapting self-report based indices (e.g. the six frequent chronic conditions described by Haagsma Each of the participating hospitals’ coders used the same coding standard but the potential for variable interpretation of the medical record and the coding directives remains, despite regular auditing of the hospital discharge data. Additionally, ICD-10-AM coding is done in Australia for reimbursement purposes. In other jurisdictions, where ICD-10 coding is not used for reimbursement or where systems limit the number of codes recorded, the number and distribution of codes may differ. Whether this would impact on ICD-10 based comorbidity adjustment is unknown but warrants consideration in the interpretation of the findings and for further research. Finally, this study focused on functional outcome. The possibility that the relationship between comorbidity and other outcomes such as health-related quality of life, return to work and pain differs from the relationship between comorbidity and functional status will be examined in further research using data from VOTOR.Mapping of ICD-10 codes to comorbidity indices showed that comorbidity is an important predictor of long term functional outcome following orthopaedic trauma, independent of age and injuries sustained. Adjustment for comorbidity is indicated when assessing risk-adjusted functional outcomes over time or across jurisdictions.The authors declare that they have no competing interests.All authors contributed to the conception and design and interpretation of data. BJG analysed the data for this study. BJG drafted the article and all authors reviewed it critically for important intellectual content. All authors approved the final version for submission.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/13/30/prepub


The detection of cytokeratin-19 (CK-19) mRNA-positive circulating tumor cells (CTC) before and/or after adjuvant chemotherapy in patients with operable breast cancer is associated with poor clinical outcome. Reliable prognostic markers for late disease relapse are not available. In this study we investigated the value of CTC detection during the first five years of follow-up in predicting late disease relapse.Blood was analyzed from 312 women with operable breast cancer who had not experienced disease relapse during the first two years of follow-up. A real-time reverse transcriptase polymerase chain reaction (RT-PCR) for CK-19 mRNA was used to detect CTC three months after the completion of adjuvant chemotherapy and every six months thereafter for a follow-up period of five years.P <0.001). Multivariate analysis revealed that persistently CTC-positive patients also had a shorter disease-free (P = 0.001) and overall survival (P = 0.001).Eighty patients (25.6% of the study population) remained CTC free throughout the five-year period. A change in CTC status was observed in 133 patients (42.6%); 64 patients (20.5%) with initially CK-19 mRNA-positive CTC during the first 24 months turned CTC-negative afterwards while 69 (22.1%) who were initially CTC-negative became CTC-positive. Ninety-nine patients (31.7%) remained persistently CK-19 mRNA-positive. After a median follow-up period of 107 months (range: 38 to 161 months), the persistently CTC-positive patients with either hormonal receptor positive or negative tumors, had a higher risk of late-disease relapse compared to the persistently CTC-negative patients (36.4% versus 11.2%, Persistent detection of CK-19 mRNA-positive CTC during the first five years of follow-up is associated with an increased risk of late relapse and death in patients with operable breast cancer and indicates the presence of chemo-and hormonotherapy-resistant residual disease. This prognostic evaluation may be useful when deciding on subsequent adjuvant systemic therapy. Invasive breast cancer is the most common malignancy in women, accounting for 28 percent of new cancer cases and 15 percent of cancer deaths . HoPersistent detection of CK-19 mRNA-positive CTC during the follow-up after the completion of adjuvant chemotherapy, tumor size greater than 2.0 cm, more than three involved axillary lymph nodes and postmenopausal status were significantly associated with reduced DFS and OS in the univariate analysis Table . MultivaWe provide, to our knowledge, the first clear evidence of a strong correlation between detection of CK-19 mRNA-positive CTC during follow-up and increased risk of late disease relapse and death in patients with either hormonal receptor positive or negative operable breast cancer. These findings support the role of CTC monitoring as an adjunct to standard clinical and radiographic methods in the evaluation of disease status during follow-up.Although the prognostic role of DTC for disease relapse and death in early breast cancer is clearly documented , the assThe present study was designed to investigate the prognostic value of CTC detection during follow-up in predicting the risk of late disease relapse. Accordingly, patients who experienced disease relapse during the first two years from diagnosis were excluded from this analysis and the median follow-up period was extended to 107 months. The changes in CK-19 mRNA-positive CTC status were thus analyzed in 312 patients and four groups were distinguished. The first group included patients without detectable CTC throughout the follow-up period, only 11.2% of whom experienced disease relapse. The second group included patients with CK-19 mRNA-positive CTC during the first two years. These patients had similar relapse risk to the persistently negative patients and might indeed have derived a benefit from the adjuvant therapy. In the third group patients with detectable CTC after, but not during, the first two years were included. The relapse risk for these patients was almost 50% higher compared to the risk of persistently negative patients, presumably due to the growth of therapy-resistant residual disease, which could not be detected early on by our method. Finally, in the fourth group patients with CK-19 mRNA-positive CTC throughout the follow-up period were included. One third of them experienced disease relapse, while one out of four patients died. This persistently positive group was by far the group with the highest relapse risk.Almost 40% of the "positive at any time" patients had detectable CK-19 mRNA-positive CTC only in one single sample. This observation could theoretically be attributed at least in part to false positive results. However, given the very low false positive rate of our assay (approximately 2%) this occurrence should be rather limited.Our results could be explained by the hypothesis that drug-resistant cancer cell clones generated during tumor evolution, constitute the re-emerging dominant tumor cell population and may start proliferating under the selective pressure of drug exposure. The high probability of subsequent disease relapse indicates that these resistant cells have a proliferative and survival advantage. This hypothesis seems to be supported by the observation (unpublished data) that the vast majority of CTC detected during the follow-up period from CK-19 mRNA persistently positive patients who had experienced disease relapse, did not express the M30 antigen which is a neo-epitope expressed only after caspase cleavage of cytokeratin 18 during early apoptosis ,25.An algorithm for the optimal timing of CTC detection during follow-up is currently lacking. Our findings suggest that serial assessments every six months for up to five years represent an acceptable timetable. Furthermore, for hormone receptor positive patients, in whom half of the recurrences occur beyond the five years, extension of the CTC serial assessments for even longer might be reasonable. These patients may represent the group which could derive benefit from an extended adjuvant treatment approach or switch to another agent.® system , which has been approved by the Food and Drug Administration for monitoring CTC in the metastatic breast cancer setting, has gained considerable attention [® system, has been evaluated in the SUCCESS trial. According to the most updated results, detection of at least one CTC in 23 mL of peripheral blood after surgical resection of the primary tumor and before the start of adjuvant systemic treatment was an independent predictor for worse DFS and OS in multivariate analysis [®. On the other hand, the ability for direct enumeration, morphological analysis and isolation of CTC for further analysis are important advantages of the latter [The QRT-PCR used in our study is not the only available assay for CTCs' detection. A semi-automated approach, the CellSearchttention ,27. The analysis . The proanalysis , as welle latter .® system) after one cycle of first-line chemotherapy will benefit from a switch to a different chemotherapeutic regimen (SWOG protocol S0500). However, for patients with operable breast cancer the lower CTC detection rate post-chemotherapy makes this strategy far more challenging [Our study has some potential limitations that should be taken into account when considering the results. This is a single institution study and the analysis was performed in one laboratory. Therefore, before the establishment of our assay as a clinically relevant test, sample analysis must be performed in several laboratories and stability during shipment must be demonstrated. Also and despite the fact that our assay has been validated in multiple cohorts and data analyses ,14,15,17llenging ,30.Additionally, the patients in our study received various types of adjuvant therapy based on available clinical and disease data at the time of enrolment. This heterogeneity may be a confounding variable, but the similarity between our findings on relapses (20.2%) and those published by the Early Breast Cancer Trialists' Collaborative Group is encouraging . FinallyOur data support a prognostic role and potential clinical utility of monitoring CTC in conjunction with standard surveillance strategies for the follow-up of patients with operable breast cancer. Given their independent unfavorable prognostic value for reduced DFS and OS, the detection of CTC after therapy could be considered as indirect evidence for the presence of chemotherapy and hormonal therapy resistant disease. Analyzing a 20-mL blood sample at various time points during follow-up might, therefore, enable clinicians to assess the efficacy of administered adjuvant therapy, limit patient exposure to ineffective agents with unnecessary toxicity, assist in the identification of patients who are most likely to benefit from clinical trials of novel therapeutics and perhaps make eradication of cancer cells more feasible, when the tumor burden is still low and before the appearance of clinically overt metastases. Since only one-third of patients with persistent CK-19 mRNA-positive CTC experience disease relapse, additional prognostic markers are needed to define better those patients who indeed might benefit from novel extended adjuvant therapies. These hypotheses can be addressed only in the context of well-designed, adequately powered, prospective, randomized clinical studies. In this way, definitive proof will be provided as to whether monitoring of the CTC can be used to improve clinical outcome in patients with operable breast cancer.cDNA: complementary deoxyribonucleic acid; CK-19: cytokeratin-19; CTC: circulating tumor cells; DFS: disease-free survival; DNA: deoxyribonucleic acid; DTC: disseminated tumor cells; ER: estrogen receptor; mL: milliliters; mRNA: messenger ribonucleic acid; OS: overall survival; PR: progesterone receptor; RT-PCR: real-time reverse transcription-polymerase chain reaction.The authors declare that they have no competing interests.ES participated in the design of the study, collected the data, analyzed the results and drafted the manuscript. MP, SA and GK: performed the laboratory work. KK, AK and SAg took care of the patients. AX performed the statistical analysis. VG and DM conceived and supervised the study and reviewed the manuscript. All authors have read and approved the final manuscript.Supplemental material. Word document containing Supplementary Tables S1 and S2 and Supplementary Figures S1 and S2.Click here for file


Clinical guidelines suggest neoadjuvant cisplatin-based chemotherapy prior to cystectomy in the setting of muscle-invasive bladder cancer (MIBC). A creatinine clearance (CrCl) >60 mL/min is frequently used to characterize cisplatin-eligible patients, and use of the CKD-EPI equation to estimate CrCl has been advocated. From a prospectively maintained institutional database, patients with MIBC who received cystectomy were identified and clinicopathologic information was ascertained. CrCl prior to surgery was computed using three equations: (1) Cockcroft-Gault (CG), (2) CKD-EPI, and (3) MDRD. The primary objective was to determine if the CG and CKD-EPI equations identified a different proportion of patients who were cisplatin-eligible, based on an estimated CrCl of >60 mL/min. Cisplatin-eligibility was also assessed in subsets based on age, CCI score and race. Actuarial rates of neoadjuvant cisplatin-based chemotherapy use were also reported. Of 126 patients, 70% and 71% of patients were found to be cisplatin-eligible by the CKD-EPI and CG equations, respectively (P = 0.9). The MDRD did not result in significantly different characterization of cisplatin-eligibility as compared to the CKD-EPI and CG equations. In the subset of patients age >80, the CKD-EPI equation identified a much smaller proportion of cisplatin-eligible patients (25%) as compared to the CG equation (50%) or the MDRD equation (63%). Only 34 patients (27%) received neoadjuvant cisplatin-based chemotherapy. Of the 92 patients who did not receive neoadjuvant chemotherapy, 64% had a CrCl >60 mL/min by CG. In contrast to previous reports, the CKD-EPI equation does not appear to characterize a broader span of patients as cisplatin-eligible. Older patients (age >80) may less frequently be characterized as cisplatin-eligible by CKD-EPI. The discordance between actual rates of neoadjuvant chemotherapy use and rates of cisplatin eligibility suggest that other factors  may guide clinical decision-making. Alternatives to neoadjuvant MVAC include dose-dense MVAC , gemcitabine-cisplatin  and cisplatin/methotrexate/vinblastine .Several therapeutic options are available to patients with muscle-invasive bladder cancer (MIBC). For patients who opt to receive radical cystectomy, current guidelines strongly recommend consideration of neoadjuvant cisplatin-based chemotherapy.Common to present neoadjuvant chemotherapy regimens for MIBC is the inclusion of cisplatin chemotherapy. Cisplatin has been used for the systemic management of bladder cancer for over three decades, and the nephrotoxicity associated with it has been well documented.et al have recently examined the Cockcroft-Gault and CKD-EPI equations in a series of 116 patients with bladder cancer treated at a single institution.Methods for calculation of estimated CrCl vary. Although the Cockcroft-Gault equation has traditionally been employed, the reduced accuracy of this equation in certain populations  has long been recognized.Patient data was obtained from an institutional bladder cancer database through the City of Hope Institutional Review Board approved protocol (IRB 12131). This database has been prospectively maintained from 1995 onwards with de-identified patient information. Prior to 2003, cases were primarily performed using a traditional open approach, whereas subsequent to 2003, cases were performed using robotic-assisted laparoscopic techniques. The database contains extensive clinical and demographic data, including age, race, gender, and Charlson comorbidity index (CCI). CCI is a system that assigns points for comorbidities and when added together can be used to predict mortality. Patients were included in the current study if they were noted to have muscle-invasive urothelial carcinoma of the bladder  are recorded at the time of the initial patient encounter . These values were used to calculate CrCl using the subsequently defined methodology.Standard methods were used to generate the estimates of CrCl using the Cockroft-Gault, CKD-EPI, and MDRD equations. Specifically, the Cockcroft-Gault equation used was as follows:t-test for categorical and continuous data, respectively. As a secondary endpoint, the proportion of patients deemed cisplatin-eligible by the MDRD equation was compared to the proportion derived from the CKD-EPI equation. Cisplatin-eligibility was also assessed in subsets based on age, CCI score and race.Demographic, clinicopathologic information and clinical outcomes data were reported, with subgroups based on actuarial receipt of neoadjuvant cisplatin-based chemotherapy. The primary endpoint of this retrospective analysis was to determine if the Cockroft-Gault and CKD-EPI equations identified different proportions of cisplatin-eligible patients amongst patients with MIBC who received cystectomy. Cisplatin eligibility was defined as a CrCl of 60 mL/min. Effect of calculation method on eligibility proportions was compared using a one way ANOVA test. Testing for differences in demographic or clinicopathologic variables between patient subgroups  and 2 waA total of 126 patients were identified who had (1) a diagnosis of MIBC, (2) had received cystectomy at City of Hope, and (3) had sufficient data available to assess renal function by the three proposed methods. The majority of patients were male (83%). A total of 34 patients (27%) received neoadjuvant chemotherapy. Of these 34 patients, 29 patients (85%) had a CrCl >60 mL/min. Interestingly, amongst patients who did not receive chemotherapy (n = 92), the majority (64%) also had a CrCl >60 mL/min. As noted in As summarized in As previously noted, cisplatin eligibility was defined by a calculated CrCl of >60 mL/min. The primary objective of the study was to determine if the CKD-EPI equation identified a greater proportion of patients to be cisplatin-eligible as compared to the Cockcroft-Gault equation, to support previously published studies.Analyses were subsequently performed, assessing cisplatin eligibility using the same three equations in subsets divided by age, race and CCI score. No differences in the proportion of cisplatin-eligibility were seen in patients <60, 60–70 or 70–80 years of age . Howeveret al.In the current study, we could not replicate the results reported previously by Tsao et al, reports such as these can only be characterized as hypothesis generating. However, we do take the added step of characterizing actuarial use of neoadjuvant chemotherapy in our cohort, which was comprised entirely of patients with MIBC . As noted in As also acknowledged by Tsao Several limitations of the study should be noted. First, comparisons are offered between three equations frequently used to estimate CrCl. However, none of these equations represents a “gold standard” – ideally, comparisons would be made to measured CrCl. It has been suggested that the measured CrCl may be the optimal means by which to discern appropriately discern candidates for cisplatin therapy.et al have proposed a consensus definition to identify patients with metastatic urothelial carcinoma “unfit” for cisplatin chemotherapy.Galsky


Small RNAs (19-24 nt) are key regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in eukaryotes. Current studies have demonstrated that microRNAs (miRNAs) act in several plant pathways associated with tissue proliferation, differentiation, and development and in response to abiotic and biotic stresses. In order to identify new miRNAs in soybean and to verify those that are possibly water deficit and rust-stress regulated, eight libraries of small RNAs were constructed and submitted to Solexa sequencing.The libraries were developed from drought-sensitive and tolerant seedlings and rust-susceptible and resistant soybeans with or without stressors. Sequencing the library and subsequent analyses detected 256 miRNAs. From this total, we identified 24 families of novel miRNAs that had not been reported before, six families of conserved miRNAs that exist in other plants species, and 22 families previously reported in soybean. We also observed the presence of several isomiRNAs during our analyses. To validate novel miRNAs, we performed RT-qPCR across the eight different libraries. Among the 11 miRNAs analyzed, all showed different expression profiles during biotic and abiotic stresses to soybean. The majority of miRNAs were up-regulated during water deficit stress in the sensitive plants. However, for the tolerant genotype, most of the miRNAs were down regulated. The pattern of miRNAs expression was also different for the distinct genotypes submitted to the pathogen stress. Most miRNAs were down regulated during the fungus infection in the susceptible genotype; however, in the resistant genotype, most miRNAs did not vary during rust attack. A prediction of the putative targets was carried out for conserved and novel miRNAs families.Validation of our results with quantitative RT-qPCR revealed that Solexa sequencing is a powerful tool for miRNA discovery. The identification of differentially expressed plant miRNAs provides molecular evidence for the possible involvement of miRNAs in the process of water deficit- and rust-stress responses. Small, non-coding RNAs have been characterized in plants as important factors involved in gene expression regulation in developmental processes ,2, as weMIRNA genes are transcribed by RNA polymerase II enzymes (Pol II) generating primary miRNA (pri-miRNA). The pri-miRNA forms an imperfect fold-back structure, which is processed into a stem-loop precursor (pre-miRNA) by nuclear RNaseIII-like enzymes called DICER-LIKE proteins  [In plants, ., DCL1) . The res., DCL1) ,9. The s., DCL1) . They ar., DCL1) . IsomiRN., DCL1) -12.Arabidopsis thaliana [http://www.sanger.ac.uk/cgi-bin/Rfam/mirna/browse.pl) [The first plant miRNAs were described in thaliana ,14 and lowse.pl) -18. Seveowse.pl) ,20, phytowse.pl) , floweriowse.pl)  and respowse.pl) ,23-25.Glycine max (L.) Merrill), the major legume crop worldwide, Subramanian et al. in 2008 [in silico screening of soybean EST databases and quantitative PCR to provide evidence for 69 miRNAs belonging to 33 families. A second study involving miRNAs and soybean root nodules was performed by Wang and colleagues [Glycine soja) was also reported by Chen et al. [In soybean ; however, none of these miRNAs were associated with water deficit or ASR stress conditions. We consider that the identification of these miRNAs is important to understanding small RNA-mediated gene regulation in soybean roots under water deficit stress and in leaves during rust infection. In this context, our goal was to identify new miRNAs and to discover those that may be regulated by water deficit and soybean rust stress. Using high-throughput sequencing, we constructed four libraries of small RNAs from the roots of drought-sensitive and tolerant seedlings in response to control or water deficit conditions. We also constructed four libraries from leaves of rust-susceptible and resistant seedlings with mock and infected conditions. A set of eight small RNAs libraries was analyzed from soybean plants. A total of 256 miRNAs were detected in Solexa sequencing. We discovered 24 novel miRNAs families and also detected several isomiRNAs in soybean. In our RT-qPCR analysis, we verified that the expression profile of several miRNAs varied during abiotic and biotic stresses. This study has important implications for gene regulation under water deficit and pathogen-infection conditions and also contributes significantly to increase the number of identified miRNAs in soybean.Currently, there are 203 miRNAs identified in Glycine max (L.) Merrill) cultivars 'Embrapa 48' as a drought-tolerant standard and 'BR 16' as a sensitive standard [3 μmoles m-2 s-1, equivalent to 8.93 × 104 lux) for a 12 h day. After 15 days, seedlings with the first trifoliate leaf fully developed  [For water deficit treatment, we used the soybean (standard . Plants l stage)  were subl stage) . The nutPhakopsora pachyrhizi collected from soybean fields in the state of Mato Grosso, which were maintained for over 10 generations on the susceptible cv. BRSMS-Bacuri. ASR identification was confirmed by ITS-sequencing analysis as described by Silva et al. [Phakopsora pachyrhizi infection. The 'PI561356' genotype was used as the resistant host, which carries an ASR resistance gene mapped onto linkage group G  and develops a reddish-brown (RB) lesion with few or no spores.The ASR reaction was evaluated in soybean plants in a greenhouse at Embrapa-Soybean  using a field population of a et al. , and it 5 spores/mL. This spore suspension was sprayed onto three plants per pot at the V2 to V3 growth stages [Urediniospores were collected from infected BRSMS-Bacuri plants in a separate greenhouse by tapping infected leaves over a plastic tray. The urediniospores were then diluted in distilled water with 0.05% Tween-20 to a final concentration of 3 × 10h stages . A solutFor the rust-stress assay, we constructed the other four libraries of small RNAs from leaves which were compounded by: 1) leaves of rust-susceptible seedlings with mock inoculation (Rust-Susceptible Leaf Control (RSLC)); 2) leaves of rust-susceptible seedlings with rust-spore inoculation (Rust-Susceptible Leaf Treated (RSLT)); 3) leaves of rust-resistant seedlings with mock inoculation (Rust-Resistant Leaf Control (RRLC)); and 4) leaves of rust-resistant seedlings with rust-spore inoculation (Rust-Resistant Leaf Treated (RRLT)).Total RNA was isolated from fresh leaves and root materials using Trizol , and the RNA quality was evaluated by electrophoresis on a 1% agarose gel. The amount of the RNA was verified using a Quibit fluorometer and Quant-iT RNA assay kit according to the manufacturer's instructions . Total RNA ( > 10 μg) was sent to Fasteris Life Sciences SA  for processing and sequencing using Solexa technology on the Illumina Genome Analyzer GAII. The libraries were constructed from the eight bar-coded samples  sequenced in a total of two channels. Quality scores were generated from Illumina's data analysis pipeline, which are similar to SAGE Phred scores with a maximum value of 40. Quality scores are based on the relative confidence of base calls using elements of cluster generation and image quality. Briefly, the processing by Illumina for the miRNA analyses consisted of the following successive steps: acrylamide gel purification of the RNA bands corresponding to the size range 20-30 nt, ligation of the 3' and 5' adapters to the RNA in two separate subsequent steps each followed by acrylamide gel purification, (3) cDNA synthesis followed by acrylamide gel purification, and a final step of PCR amplification to generate cDNA colonies template library for Illumina sequencing. After removing the adapter sequences, the sequences were trimmed into different read lengths from 19 to 24 nt for further analysis.http://www.phytozome.net) assembly using the SOAP program [The reads were grouped into unique sequences, and the read counts were calculated for each library. The sequences that presented low read counts (read count < = 2) were discarded from the final list of unique sequences, which are referred to as a tag. The sequences were mapped into the soybean genome  the  program . If a pe2, 0.25 U Platinum Taq DNA Polymerase (Invitrogen) and 200 nM of each reverse and forward primer. The universal reverse primer (5' GTGCAGGGTCCGAGGT 3') was used in all RT-qPCR reactions. Samples were analyzed in biological triplicate in a 96-well plate, and a no-template control was included. We used MIR156b (5'- TGACAGAAGAGAGAGAGCACA - 3'), MIR172ab (5'- AGAATCTTGATGATGCTGCAT - 3') and MIR1520d (5'- ATCAGAACATGACACGTGACAA - 3') as reference genes, which has been demonstrated as optimal normalizers for water deficit and rust-stress analysis in Glycine max [-ΔΔCt method. Student's t-test was performed to compare pair-wise differences in expression. The parameters of two-tailed distribution and two samples assuming unequal variances were established. The means were considered significantly different when P < 0.05.To validate predicted new miRNAs, RT-qPCR in respect to eleven miRNAs was performed to examine their expression across the eight different libraries. From those, six were new miRNAs belonging to conserved soybean miRNAs families ; one new miRNA pertencing to conserved miRNAs families in other plants species (MIR397ab); and four were miRNAs belonging to novel miRNAs families . The forward miRNAs primers were designed based on the full miRNAs sequence, and the reverse primer was the universal reverse primer for miRNA . The stecine max . The conhttp://biocomp5.noble.org/psRNATarget/) [Glycine max database (DFCI gene index release 15). The total scoring for an alignment was calculated based on the miRNA length, and the sequences were considered to be miRNA targets if the total score were less than 3.0 points (mismatch = 1 and G:U = 0.5). Results from these analyses were individually inspected on the Phytozome, where the loci and protein annotation were obtained. In order to look for evidences of the predicted targets of the novel identified miRNA, we searched for the miRNA targets sites in the soybean degradome libraries published by Song et al. [Target prediction for miRNAs is straightforward because it is assumed that most of them match their targets with almost perfect complementarity ,9. The pTarget/)  which cag et al.  availablg et al. .To identify miRNAs from soybean under water deficit and rust stresses, we generated eight libraries of small RNAs species. From these libraries, a total of 256 miRNAs ranged from 19 to 24 nt-long sequence sizes were identified  are a population of known miRNA variants. They are caused by an imprecise or alternative cleavage of Dicer during pre-miRNA processing . We deteThe stem-loop RT-qPCR was used to validate and measure the expression of the respective miRNAs: MIR166a-5p, MIR166f, MIR169f-3p, MIR397ab, MIR482bd-3p, MIR1513c, MIR4415b, MIR-Seq07, MIR-Seq11, MIR-Seq13 and MIR-Seq15ab, detected by Solexa sequencing. These miRNAs were validated in all genotypes analyzed during dehydration and rust stress. The relative expressions of these miRNAs in the same eight conditions are shown in Figure MIR166-5p, MIR169f-3p, MIR1513c, MIR397ab and MIR-Seq13) presented the same behavior during the water deficit stress. These miRNAs were commonly up-regulated during the stress condition in the sensitive genotype, and the opposite occur in the tolerant genotype, where they were down-regulated during the water deficit. MIR-Seq11 and MIR-Seq15 demonstrated a similar expression across the four conditions. Water deficit significantly increased MIR-Seq11 and MIR-Seq15 expression in the roots compared to the control condition in the sensitive genotype, but both miRNAs did not vary in the tolerant plants. MIR166f had its level increased in the sensitive genotype and decreased in the tolerant during the stress compared to the control situation. Interestingly, both genotypes presented the same level during the control condition. In the sensitive plants, MIR-482bd-3p showed a strong decrease when submitted to water deficit, being this low level equally observed in the tolerant genotype during the control condition and decreasing when subjected to stress. MIR4415b presented an effective rise in its expression level during the water deficit in the sensitive plants, and its high level was also observed in the tolerant genotype independent of the condition. Both sensitive and tolerant genotype exhibited the same expression pattern for MIR-Seq07 and its level was increased during the stress compared to the control situation.To identify water deficit-responsive miRNAs, we compared the expression profiles of the 11 miRNAs in both genotypes before and after stress Figure . A set oMIR166a-5p, MIR166f, MIR169-3p, MIR397ab and MIR-Seq13 were dow-regulated in the susceptible genotype during pathogen infection, and equally expressed in the resistant plants. The level of MIR482bd-3p did not vary significantly between the two different conditions in the susceptible. However in the resistant genotype, its level is higher during the control condition and decrease with the pathogen attack. MIR1513c presented unchangeable expression in the control and stressed condition for both genotypes, but when we compared the two genotypes; the resistant was down-regulated compared to the susceptible. A strong decrease was observed for MIR4415b in the rust infection when compared with the control in the susceptible plants, and its level is higher in the resistant genotype showing no expression alteration between the conditions. MIR-Seq07 was down-regulated with respect to the soybean rust infection in both genotypes. Significant difference was observed in MIR-Seq11 expression between the mock and infected plants from the susceptible genotypes. This miRNA presented a low expression level after rust inoculation, and its level decreased in the resistant genotypes remaining similar in the both conditions. MIR-Seq15ab expression level was significantly decreased in the rust compared to the mock treatment in the susceptible genotype, the opposite occurs in the resistant genotype, when the control showed a lower level of expression compared to the stressed condition.The RT-qPCR analyses of four libraries from the rust assays are shown in Figure MiRNAs suppress gene expression by inhibiting translation, promoting mRNA decay or both . Target Glycine max peroxidase precursors mRNAs as potential targets. The oxidoreductase and a transcription regulator factor were predicted to be targeted by MIR-Seq13; and for the MIR-Seq15 only a translation initiator factor was predicted as a target.Among the 24 novel identified miRNAs families, only 14 families had targets predicted Table . The miRAfter a comparative analysis of our novel identified miRNAs and the degradome libraries of developing soybean seeds it was possible to identify specific sequences in the degradome that corresponds to the downstream sequence of the predicted miRNA recognition site. We identified target sequences to six among the 24 novel soybean miRNAs . The list of the 10 identified genes is composed by a glucosyl transferase, serine carboxypeptidase, fructose biphosphate aldolase, three leucine-rich repeat protein, two peroxidases and two ATP dependent RNA helicases  . Among tGlycine max. These miRNAs were identified from eight different libraries from precursors with stem-loop secondary structures that also map to the soybean genome  were very common in our population of small, detected RNAs. Out of the isomiRNAs, we observed 21 pairs of sense and antisense miRNAs. The duplex presents the antisense strand paired to the corresponding miRNA with two nucleotides 3' overhangs  did not vary across the mock and rust infection. Surprisingly, MIR-Seq07 was the unique miRNA that was down-regulated during the fungi infection for both genotypes analyzed in our study. We already mentioned that the MIR-Seq07 had predicted protein target related to metabolism and thus its possible association with water stress. However MIR-Seq07 also had predicted LRRs (leucine-rich repeats)-domain target which are known to be present in disease resistance proteins ,60. ThisThe present study detected a large number of small RNA sequences that were characterized as novel and as already known soybean miRNAs. We grouped some of these unique sequences into 24 novel soybean miRNAs and further classified several of new members in known families or as new loci in the soybean genome. Validation of new miRNA with quantitative RT-qPCR revealed that Solexa sequencing is a powerful tool for miRNA discovery. Many miRNA expression patterns were up- or down-regulated by water deficit and rust stresses, which is an important discovery. Future investigations should use supplementary experimental approaches to verify the targets and to understand the complex gene regulatory network of these miRNAs. This work will contribute to improve systems to support soybean crop production and to mitigating crop losses during biotic or abiotic stresses.FRK and RM conceived and designed the study. FRK performed the sequence analyses to identify the miRNAs and secondary structures and to predict the target genes conceived, executed the RT-qPCR, performed the data management and processing, and wrote the draft manuscript. RM was the supervisor of this study, provided critical revision, obtained financial support and performed data interpretation. LFVO contributed to the data assembly, prediction and identification de new miRNAs. LM and MA contributed to the analysis of the miRNA secondary structures and processing of the data. FR, JM, JFB and RSM performed the plant experiments and RNA extractions. ALN, FCMG and RVA provided the studied material, critically revised the article for important intellectual content and obtained funding. MFC, GAGP and LCN created the Perl scripts to identify the microRNAs. MFC participated in writing the methods section. All authors read and approved the final version of manuscript.Predicted precursor structures of all miRNAs identified. The mature miRNAs (red) and pre-miRNA sequences with chromosome and locus information. The pre-miRNA length (nt) and its directional information (sense (+) or anti-sense (-) compared to the soybean genome sequence) is provided. The fold-back structure with respect to the free energy value (dG) was predicted using the Mfold program.Click here for fileIdentified targets of known conserved plant miRNAs families. a The Data from Phytozome version 6.0. b Pairing obtained in psRNATarget Server: "|" indicates a Watson-Crick base pairing; ":" is a G:U base pairing, and "-"indicates a mismatch.Click here for fileThe soybean transcript loci which were identified as new-miRNA families target by degradome sequencing. The miRNA target site is indicated in red and underlined while the degradome sequence is highlighted.Click here for file