Despite advances in surgery and adjuvant regimes, gastrointestinal malignancy remains a major cause of neoplastic mortality. Immunotherapy is an emerging and now successful treatment modality for numerous cancers that relies on the manipulation of the immune system and its effector functions to eradicate tumour cells. The discovery that the pan-epithelial homotypic cell adhesion molecule EpCAM is differentially expressed on gastrointestinal tumours has made this a viable target for immunotherapy. Clinical trials using naked anti EpCAM antibody, immunoconjugates, anti-idiotypic and dendritic cell vaccines have met variable success. The murine IgG2a Edrecolomab was shown to reduce mortality and morbidity at a level slightly lower than treatment with 5FU and Levamisole when administered to patients with advanced colorectal carcinoma in a large randomised controlled trial. Fully human and trifunctional antibodies that specifically recruit CD3-positive lymphocytes are now being tested clinically in the treatment of minimal residual disease and ascites. Although clinical trials are in their infancy, the future may bring forth an EpCAM mediated approach for the effective activation and harnessing of the immune system to destroy a pathological aberrance that has otherwise largely escaped its attention. Gastrointestinal malignancy remains one of the largest causes of neoplastic mortality and morbidity. The use of immunotherapy to target tumours is well established. This combines antibodies that specifically target antigens selectively expressed on tumour cells and cytotoxic effector mechanisms. Successful clinical use includes the treatment of breast cancer by HER-2-specific trastuzumab , follicuImmunotherapeutic strategies for abdominal malignancy include targeting of the cell-adhesion molecule EpCAM. This article reviews the expression and function of EpCAM in normal and neoplastic tissue. The variety of strategies to target EpCAM are discussed, followed by a review of their use in clinical trials to target GI malignancy.EpCAM is a pan-epithelial differentiation antigen expressed on the basolateral surface of all carcinomas to varying degrees. As a homotypic cell adhesion molecule, it is intimately integrated within the Cadherin–Catenin and WNT pathways. It has recently been shown to modulate the expression of proto-oncogenes such as c-myc.α-, β- and γ-catenins forming the cadherin–catenin complex .cis-dimer. Dimers on opposing membranes can associate via a head-to-head interaction to form tetramers with moderate affinity consistent with reversible intercellular associations. It is not known how exactly antibody binding correlates with variable glycosylation or oligomerisation in a functional or structural sense, and further investigation is required.90% of Colorectal carcinoma cells express EpCAM but in a differential form. Modifications include variable glycosylation analogous to tumour-specific antigens such as colonic tumour antigen MUC-1 . EpCAM eTissue microarray studies allow comprehensive assessment of the tumour expression of EpCAM. A recent study compared 3900 tissues of tumour of stratified stages and grades of 134 different histological subtypes sourced from head and neck, lung, gastrointestinal, breast, urogenital and mesenchymal tumours. Seventy-five per cent tumour categories expressed EpCAM. At least weak EpCAM expression in >10% of tumours was observed in 87 of 131 different tumour categories. Colonic (81%), gastric, pancreatic (78%) and lung carcinomas revealed a high proportion of strongly positive tumours, suggesting EpCAM is an attractive target for pan-carcinoma immunotherapy  had a significantly better 10-year survival compared with patients with any loss: 42 α-catenin and β-catenin. This may be due to silencing of the inhibition that EpCAM exerts on E-cadherin. Alternatively, EpCAM gene silencing may lead to decreased cytoplasmic β-catenin through an increase in its association with the E-cadherin adhesion complex. Hence, reducing EpCAM may decrease β-catenin availability for the wnt pathway and activation of its target genes downstream . We postulate that the absence of homotypic adhesions in the vascular microenvironment may be the causal link. Indeed, EpCAM-targeted immunotherapy may be more effective in the context of established tumours or metastases as opposed to fluid-borne disease. This does not preclude ascites due to peritoneal metastases for which treatment with trifunctional antibodies is effective. However, the efficacy of destroying blood-borne circulating tumour cells in a hope of eradicating minimal residual disease may be limited.Immunotherapy manipulates competent host immune system inducing tumour growth inhibition, regression or cytolysis. Approaches include the use of monoclonal antibodies and their derivatives, hybrid bispecific  antibodies, tumour cell vaccines, anti-idiotypic antibodies and dendritic cell vaccines. Pure immunomodulatory cytokines have been used to enhance the effect of monoclonal antibodies (MAbs).in vivo remain controversial. Cytotoxic mechanisms include antibody dependent cell cytotoxicity (ADCC) mediated by natural killer cells and T lymphocytes, complement mediated cytolysis (CMC) and opsonisation promoting phagocytosis mediated by PMNs.The mechanisms by which anti-EpCAM antibodies exert tumour inhibition c-myc: this remains untested. The majority of anti-EpCAM antibodies produced are specific for epitopes within the first of two EGF-like domains in the extracellular segment of EpCAM  show substantially reduced responses to antitumour MAbs. In contrast, mice deficient in the inhibitory receptor FcγRIIb-CD32 respond to the same antibodies with enhanced ADCC and tumour growth inhibition. Engineering the Fc portion to maximise FcγRIII binding may enhance the therapeutic benefit of immunotherapeutic agents. The significance in human context remains unknown , disease recurrence (23%) and metastases leading to further phase II and III trials.In 2002, Punt published results of a trial of 2761 patients randomised to MAb 17-1A monotherapy, 5-FU and Folinic-acid or 5-FU+Ederocolomab. No additional benefit was seen by adding immunotherapy to the standard chemotherapy regimen at 26 months. Immunotherapy alone was associated with significantly shorter disease-free survival; ederecolomab was removed from circulation.The discrepancy between preclinical and clinical findings has led to much debate. What are the reasons for this discrepancy?EpCAM expression density varies at different stages of tumour growth suggesting that patient antigen positivity should be assessed before clinical use. EpCAM density is a proven predictor of survival in breast cancer patients  and compatibility of syngeneic effector cells -α. Il-2 stimulate a humoral and cell-mediated immune response. The cell-mediated response causes CD8+ Cytotoxic T lymphocytes proliferation and subsequently mature under the influence of IFN-γ and TNF-α to mediate cytolysis either by Fas–Fas ligand interaction to cause apoptosis or by the exocytosis of vesicles containing perforins and other proteases.Anti-idiotypic antibodies attempt to stimulate a prolonged, active immune response. Immunisation with a primary antigen such as EpCAM generates anti-EpCAM antibodies termed Ab1. Anti-idiotypic antibodies are then generated against Ab1. These antibodies termed Ab2 mimic the tertiary structure of EpCAM. Ab2 antibodies are used as surrogate immunogens for the production of Ab3, which are active against the original antigen EpCAM. Ab2 are endocytosed by APCs and hence represented by major histocompatibility complex class II antigens to stimulate CD4+ T helper cells. This occurs in the presence of the co-stimulatory molecule CD80/86 on APCs interacting with CD28 on TH cells. These then proliferate and mature to lymphocytes secreting cytokines Il-2, IFN-Although the prolonged spectre of active immunity is attractive in theory, multiple trials assessing the efficacy of anti-idiotypic antibodies against EpCAM for advanced GI cancer have shown only marginal success .Among the first was an anti-idiotypic antibody targeted against the GA733-antigen associated with EpCAM (Co17-1A). Antigen-specific T-cell immunity was detected in all six patients immunized  prepared as alum precipitates. The majority produced Ab3 antibodies sharing epitopes with the corresponding Ab1. One out of three displayed delayed-type hypersensitivity and specific T cells that readily proliferated in the presence of antigen et al, 2002).The immunogenicity of full-length EpCAM encoded by recombinant vaccinia virus (VV GA733-2) has been assessed in a murine model. This induces antibodies to previously unknown epitopes and promotes ADCC of CRC targets with murine macrophages. Immunized mice developed Ag-specific, proliferative and delayed-type hypersensitive lymphocytes. The proliferation of ras-transformed syngeneic tumour cells expressing human EpCAM was suppressed in vivo suggesting the potential of VV GA733-2 as a candidate vaccine for patients with CRC  such as macrophages. These are described as being bifunctional and monospecific. In trifunctional antibodies, the two halves of the FAb segment have different specificity: they are bispecific and trifunctional.Normal IgG molecules compose of Fc and FAb segments . The monγR binding to the Fc portion of the antibody. This antibody consists of a Murine IgG2a associated with an anti-EpCAM FAb segment and Rat IgG2b associated with anti-CD3  successfully constructed a bispecific/trifunctional, targeting both EpCAM and CD3 (BiUII or Removab). The rationale being that ADCC is complemented by the presence of CD3+ T-lymphocytes in addition to macrophage/monocytes, NK and dendritic cells, known to express Fcin vitro experiments of ADCC with squamous cell carcinoma cell lines, effector cells and BiUII demonstrated increased production of IL-1β, IL-2, IL-6, IL-12 and DC-CK1. It is postulated that simultaneous stimulation of accessory cells and T lymphocytes leads to antigen presentation to T-lymphocytes inducing immunomodulation and cytotoxicity. BiUII induces production of IL-2 in the presence of EpCAM+ cells activating accessory and T cells without the usual requirement of exogenous IL-2. It is thought that an immunologically self-supporting tri-cell complex is formed, which is efficient for immune cell activation.Initial Interestingly, computerised sequential video microscopy shows that cytolysis occurs within 1–3 days, with no difference between patient and healthy donor PBMCs. The mode of cell death is characteristically necrotic and not apoptotic. Lymphocytes with pore-forming perforin proteins surround the tumour cells causing cytolysis  and all tumour controls died by day 28. BiLu-treated surviving animals displayed an IgG2a humoral immune response but no IgG1 response. Nonsurvivors mounted only weak immune responses. Survivors rechallenged with reduced but potentially lethal doses of transfected cells established long-lasting immunity, actively rejecting tumour at day 144.All BiLu-treated animals survived; those treated with parental antibodies had significantly lower survival , showing that Removab was safe and effectively reduced ascitic flow and tumour-cell content. A substantial phase II/III trial assessing efficacy in patients with all causes of malignant ascites including primary gastrointestinal tumours commenced in September 2004 (250 patients), and a Phase IIa study of platinum refractory ovarian cancer patients is also underway. Finally, a Phase I/II study of patients with peritoneal carcinomatosis owing to GI tumours but without symptomatic ascites is underway. Preliminary results are promising, indicating the utility of Removab in the treatment of minimal fluid borne disease and micrometastases.The discovery of antigens differentially expressed either on the surface of tumour cells or on part of their metabolic repertoire has created an opportunity for a new modality of cancer treatment. The concurrent development of monoclonal antibody technology has realised the potential of this opportunity.Immunotherapy has the potential not only to provide magic bullets that trigger changes in the trajectory of cellular differentiation but also to harness with exquisite specificity the devastating storm of the immune system to eradicate tumours. Its potential for a long-lasting memory can also reduce recurrence.The results of the largest clinical immunotherapy trials targeting EpCAM highlighted discrepancy between the high level of ADCC seen in vitro and disease-free survival and mortality. The classic problems of patient selection, time of therapy, delivery and specificity require further investigation.in vitro.Development of fully human MT201 antibody: currently being tested in phase I and II trials attempts to optimise delivery by alleviating HAMA production. Alternative approaches involving removal of the Fc portion retaining the Fab, F (ab)2 have been studied but only appear to be useful The functionally intricate role of EpCAM within the WNT pathway remains largely unexplored and therapeutically unexploited.The temporal expression of EpCAM in the life cycle of human gastrointestinal tumours remains largely unstudied. Indeed, there may be a golden window of opportunity when EpCAM expression increases to a point after which metastatic tumour cells reduce expression. Use of EpCAM targeted immunotherapy earlier in disease progression may enhance tumour penetration and delivery to target cells in solid tumours.in vivo and therefore we can only marginally improve EpCAM-based immunotherapy. Full structural information correlating the effect of antibodies on EpCAM structure, function and downstream pathways as well as gene regulation and proliferation require further research. Although it is widely held that ADCC is the major mechanism of tumour inhibition direct antibody effects in vivo cannot be precluded.Until we understand exactly how EpCAM antibodies on cancer cells, we cannot fully grasp what really happens Through greater understanding of the Fc/FcR interplay of antibody and effector cells in cancer patients, it may be possible to engineer Fc portions of trifunctional antibodies with greater accessory cells specificity maximising ADCC.Pre-clinical efficacy of bispecific/trifunctional antibody immunotherapy is proven. Utilisation of these exciting new antibodies to treat human solid carcinomas remains untested. The significance of human clearance of rodent antibody in the case of Removab remains unknown.Clinical trials in this area are in their infancy. Preliminary results from intraperitoneal treatment of malignant ascites are promising. Indeed, the future seems exciting and may bring forth an EpCAM-mediated approach for the effective activation and harnessing of the immune system to destroy a pathological aberrance that has otherwise largely escaped attention.


Anaerobic glycolysis of the tumour cells was unaltered by hyperthermia. Respiration and anaerobic glycolysis of normal rabbit liver, kidney and red blood cells were unaffected by the elevated temperature. Local heat was applied to established VX2 tumours in vivo, with a subsequent reduction in tumour size in all cases, the most effective therapy regime being 3 one-hour applications of heat within the mean cell generation time of the tumour. Following heating there was rapid and widespread tumour cell necrosis and lysis, with subsequent replacement of the tumour architecture by connective tissue. There was a prolongation of survival time in 50% of the treated animals, which are still alive 18 months after therapy; all the control animals died within 10 weeks. The selective inhibitory effects of hyperthermia on cancer cells, and its application to human neoplasms, are discussed.Hyperthermia (42° C.) exerted an inhibitory effect on the O


The comet assay was used to estimate radiobiological hypoxic fraction across a full range of tumour oxygenations in C3H mammary tumours implanted into the feet of female CDF1 mice. Tumours were either clamped before irradiation or mice were allowed to breath air, 100% oxygen, carbogen or carbon monoxide for 5-35 min before and during exposure to 15 Gy. For the alkaline comet assay, tumours were excised after irradiation and individual tumour cells were analysed for DNA single-strand breaks. Hypoxic cells were defined as those cells with approximately three times fewer single-strand breaks than aerobic cells. Radiobiological hypoxic fraction was calculated by fitting DNA damage histograms to two normal distributions, representing the response of the aerobic and hypoxic populations. The percentage of hypoxic cells estimated using the comet assay was then compared with hypoxic fraction measured using a clamped tumour control assay. Carbogen and oxygen breathing reduced the normal hypoxic fraction from 14% to 2-3% in this tumour, whereas 75-660 p.p.m. carbon monoxide progressively increased the hypoxic fraction from 18% to 82%. The slope of the line comparing the two methods was 1.23 with 95% confidence limits of 1.12-1.33 (r2 = 0.994). In the SCCVII squamous cell carcinoma growing subcutaneously in C3H mice, a similar correlation was observed between hypoxic fraction measured using the comet assay and hypoxic fraction measured in the same tumour cells using the paired survival curve assay (slope = 1.20 with 95% confidence limits of 1.03-1.37). These results confirm the ability of the comet assay to provide an accurate estimate of radiobiological hypoxic fraction over a wide range of tumour oxygenations and between two tumour types.


The effects of flavone acetic acid (FAA) on the coagulation properties of plasma from tumour-bearing and non-tumour-bearing mice have been investigated. The study was carried out primarily on CBA mice and the CaNT tumour, although substantiating data are included for two other tumours grown in the WH strain. FAA was injected at a range of single doses up to a maximum of 300 mg kg-1, and clotting properties of the plasma were measured in vitro at various times after FAA administration. Platelet numbers and the concentration of fibrin degradation products (FDP) in the plasma were also determined. Following a dose of 300 mg kg-1, the clotting times were significantly reduced at 15-30 min in both tumour-bearing and non-tumour-bearing mice of both strains. Detailed studies on coagulation in the CBA strain (+/- CaNT tumour) indicate that in tumour-bearing animals the initial decrease in clotting time is followed 4-6 h later by an increase in clotting time, thrombin time and FDP levels. Platelet counts of tumour-bearing mice also decreased significantly over this period. Similar experiments in non-tumour-bearing mice did not show these late effects. All the data from the coagulation tests on mice with CaNT tumours are consistent with the hypothesis that intravascular coagulation occurs following treatment with FAA, and that vascular occlusion in tumours, as a results of FAA-induced coagulopathy, may contribute to tumour regression.


The investigations reported in this paper aim to exploit tumour necrosis factor (TNF)-induced vascular changes in an attempt to increase the tumour uptake of specific monoclonal antibody. The vascular permeability to monoclonal antibody of a human tumour xenograft increased 2.6-fold by 1 h post injection of 2.5 x 10(3) U of TNF, although this effect was lost by 3 h. The normal tissues also demonstrated increased vascular permeability to IgG, but to a lesser extent. Liver permeability increased 1.5-fold at 1 h but returned to the control value by 6 h. Lung permeability increased 1.4-fold at 1 h post injection and returned to normal by 3 h. Muscle values were not significantly increased compared with controls. The blood activity was cleared more quickly in the TNF-treated mice . This was probably due to the increased vascular permeability in normal organs of treated mice. At 1 day and 3 days post injection, the tumour uptake of the specific, but not the control, antibody was significantly increased by 25% and 29% respectively. This resulted in an increase in the area under the tumour activity curve, and therefore tumour radiation dose, of 25% in treated compared with control mice. In addition, a consequence of the faster blood clearance of the isotope in the TNF-treated mice was a reduction in the area under the blood activity curve of 12%, thereby reducing systemic toxicity. The increase in vascular permeability to IgG following TNF injection resulted in both specific and control antibodies having improved access to the tumour antigens, and a transient increase in uptake was observed. Only in the case of the specific antibody was the increase maintained, since this antibody binds to the available antigenic sites, whereas the control antibody was cleared from the tumour without binding. No evidence of tumour necrosis was observed at the TNF doses given, nor was there any toxicity to the mice.


Sprague-Dawley rats aged 50 days were given single oral doses of the carcinogen, DMBA.Rats receiving the carcinogen at the same stage of the oestrous cycle were grouped together and mammary tumour production was compared between these groups.When the carcinogen was given during di-oestrus the mean number of tumours per animal was significantly greater than when it was given at other stages in the oestrous cycle. There was considerable variation in total tumour yield from one batch of animals to another.


A case of metastatic granulosa cell tumour of the ovary is reported. Investigations revealed a secondary tumour in segment VI and VII of the liver. Right hepatic resection was performed. Microscopic findings revealed a tumour with histological features identical to that removed eleven years before.


The relative efficiency of active nonspecific or specific immunotherapy of developing methylcholanthrene induced fibrosarcomata with C. parvum was compared. For nonspecific immunotherapy, mice were challenged with tumour cells s.c. or i.v., and 2 days later injected i.v. with dilutions of C. parvum. The only significant effect was a retardation of s.c. tumour growth by the highest concentration of C. parvum (350 mug). However, active specific immunotherapy, using mixtures of C. parvum and irradiated or living tumour cells in the footpads, suppressed tumour growth when given at 2 or 6, but not 10, days after tumour challenge. Successful therapy required: sufficient tumour cells ); an optimal dose of C. parvum ; an intact T cell system; the same tumour cells for challenge and treatment. The specificity was confirmed in a protection system in which treatment was given 7 days before tumour challenge. No protective immunity could be achieved with mixtures of C. parvum and foetal cells. Thus in this system C. parvum potentiates protective immunity only to the tumour unique TSTA.


EL4 tumour cells maintained in culture were separated by FACS analysis to Ly-6.2 negative and Ly-6.2 positive subsets. The Ly-6.2 negative subset gained expression of this determinant on repeated in vivo passage in C57BL/6 mice. Both subsets injected intraperitoneally or intramuscularly in syngeneic mice induced identical changes in lymphocyte profiles. There was generalised lymphocytolysis in both T- and B-cell compartments. The Lyt-1+, 2- T-lymphocytes were more susceptible to cytolysis causing an alteration of the proportional representation of the Lyt-2+ subset from 30% of splenic T-cells  to over 90% of remaining T-lymphocytes in tumour bearing mice. There was thymic regression in both groups of mice with a resultant thymocyte population expressing the range of phenotypes of mature medullary cells. In spite of similar rates of growth both in vivo and in vitro and identical effects on the lymphoid system the Ly-6.2 negative and Ly-6.2 positive tumour subsets were different in their metastatic potential. Mice injected intramuscularly with either subset had enlarged spleens by the second week of tumour growth caused largely by the accumulation of Ig, Lyt-1 and Thy-1 negative cells. Tumour cells were present only in the group injected with the Ly-6.2+ subset. These mice died of their tumour load a week earlier than those injected with the Ly-6.2- tumour cells.


Pleural effusions are a common clinical problem and affect about one million people in the United States and United Kingdom each year. Over 60 causes of pleural effusion have been identified; establishing the definitive aetiology can be difficult, and often requires invasive procedures. Guidelines state that macroscopic examination of the fluid should be the first step in determining the aetiology of a pleural effusion. Papillary thyroid carcinoma is an uncommon cause of malignant pleural effusion, with only 10 cases reported in the literature, their physical characteristics and composition having been rarely described. We describe for the first time a distinctive brown colour of the malignant effusion (despite centrifugation) from a rare case of metastatic papillary thyroid cancer to the pleura, associated with a high pleural fluid iodine content. Such a characteristic may be useful in expediting diagnosis of a malignant pleural effusion in the appropriate clinical context.We present the case of a 71-year-old Caucasian man with metastatic papillary thyroid cancer; a large, long-standing, right-sided pleural effusion and a 83-fold higher pleural thyroglobulin level compared to corresponding serum, supporting this malignancy as the cause of the patient’s effusion. The pleural fluid had a distinctive pigmentation similar to iodine-containing antiseptic preparations. Biopsy during medical thoracoscopy confirmed metastatic papillary thyroid carcinoma. Analysis of pleural fluid showed a pleural thyroglobulin level over 80 times that of serum levels . Pleural fluid iodine content was 23,000ug/L and may account for the fluid’s distinctive pigment, as iodine is an essential component in thyroglobulin and thyroid hormone synthesis.Pleural fluid pigmentation may aid diagnosis in the appropriate clinical setting. A distinctive iodine-like brown colour of pleural fluid may represent elevated iodine content and should raise consideration of metastatic thyroid cancer as a cause for a pleural effusion. Pleural effusion is common in clinical practice and can be caused by over 60 pulmonary or systemic disorders. Clinical guidelines  and convMalignant pleural effusions are a common clinical problem, and a wide variety of malignancies can metastasise to the pleura. Papillary thyroid carcinoma is an uncommon cause of malignant pleural effusion, with only 10 cases in total reported in the literature -9, theirWe report for the first time a distinctive dark brown pigmentation of the pleural fluid from metastatic malignant papillary thyroid carcinoma, as explained by our biochemical finding of a high iodine content in the fluid. The pleural fluid thyroglobulin was also the highest reported to date.A 71-year-old Caucasian man diagnosed with metastatic papillary thyroid cancer was referred to the Pleural Clinic for management of a large right-sided effusion. He was diagnosed with papillary thyroid cancer (PTC) 11 years prior, and was treated with total thyroidectomy and adjuvant radio-iodine treatment. Mutation analysis was not performed. Over the next four to six years of follow-up, he received further radioactive iodine therapy due to disease recurrence with rising serum thyroglobulin levels. Small pulmonary metastases were first identified as fluorodeoxyglucose (FDG)-avid lesions on positron emission tomography-computed tomography (PET-CT) five years before this presentation, together with right-sided pleural thickening. Since then serial serum thyroglobulin showed continual rises from 51 to 1100ug/L over four years. The patient had iodine-refractory disease and was enrolled in a clinical trial of pazopanib, a multi-targeted receptor tyrosine kinase inhibitor, two years prior to his presentation to the Pleural Clinic. Treatment with pazopanib, significantly reduced his thyroglobulin level to 290ug/L.He was an ex-smoker, with a history of ischaemic heart disease, peripheral vascular disease and asbestos exposure.When assessed at our Pleural Clinic, he was dyspnoeic and had a large, right-sided pleural effusion on CT scan Figure  and ultrMedical thoracoscopy revealed multiple areas of tumour deposits on the parietal and visceral pleura, clearly shown in the additional movie file , and is the highest level yet reported. Given the unusual colour of the fluid and its striking resemblance to iodine solutions, we analysed the pleural fluid total iodine content using inductively coupled plasma mass spectrometry  and showThe patient received a talc poudrage pleurodesis and continued with pazopanib therapy.We report a case of metastatic PTC to the pleura and describe for the first time a high iodine content of the fluid that explains an unusual dark brown colour of the pleural fluid not previously reported.Papillary thyroid cancer is the most common form of thyroid malignancy and metastatic disease tends to involve local lymph nodes . DistantInspection of the pleural fluid is often considered the first step in its analysis, and can provide clues to empyema, chylous effusions, haemothorax or oesophageal perforation. This is, however, the first report of a distinctive dark brown colour in a pleural effusion with a corresponding high iodine content in the fluid, presumably from synthesis of iodine-rich molecules by pleural PTC deposits. Brown effusions in themselves are not unique or pathognomic of metastatic PTC, and brownish effusions, of different color intensity, can be seen with degradation of haemoglobin, rupture of amoebic liver abscesses and parasitic infection . SimilarSerum thyroglobulin is well established as a monitoring method for disease activity in PTC . In thisThis case is the first to report high iodine content and an associated distinctive brown colour in the malignant effusion fluid from metastatic PTC. The usefulness of these findings in aiding diagnosis of metastatic pleural disease from PTC deserves investigation. In the meantime, a dark brown iodine-coloured effusion should raise the consideration of metastatic thyroid diseases.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.AR analysed and interpreted the patient data regarding the pleural disease and fluid characteristics, reviewed the literature, and provided a major contribution to the writing of the manuscript. EML provided clinical and technical advice and performed the pleural fluid chemical analysis. MM provided clinical advice and reviewed the manuscript. YCGL performed the pleursocopy, edited the manuscript and advised on all aspects of the case report. All authors read and approved the final manuscript.Right pleural space during medical pleuroscopy. The distinctive pleural fluid colour is demonstrated and lesions confirmed as metastatic papillary thyroid carcinoma shown on the parietal and visceral pleura.Click here for file


An increase of glomerular filtration rate (hyperfiltration) is an early functional change associated with type I or type II diabetes mellitus in patients and animal models. The causes underlying glomerular hyperfiltration are not entirely clear. There is evidence from studies in the streptozotocin model of diabetes in rats that an increase of proximal tubular reabsorption results in the withdrawal of a vasoconstrictor input exerted by the tubuloglomerular feedback (TGF) mechanism. In the present study, we have used micropuncture to assess single nephron function in wild type (WT) mice and in two strains of type I diabetic Ins2+/- mice in either a C57Bl/6 (Akita) or an A1AR-/- background (Akita/A1AR-/-) in which TGF is non-functional. Kidney glomerular filtration rate (GFR) of anesthetized mice was increased by 25% in Akita mice and by 52% in Akita/A1AR-/-, but did not differ between genotypes when corrected for kidney weight. Single nephron GFR (SNGFR) measured by end-proximal fluid collections averaged 11.8 ± 1 nl/min (n=17), 13.05 ± 1.1 nl/min , and 15.4 ± 0.84 nl/min  in WT, Akita, and Akita/A1AR-/- mice respectively. Proximal tubular fluid reabsorption was not different between WT and diabetic mice and correlated with SNGFR in all genotypes. We conclude that glomerular hyperfiltration is a primary event in the Akita model of type I diabetes, perhaps driven by an increased filtering surface area, and that it is ameliorated by TGF to the extent that this regulatory system is functional. The issue of diabetic hyperfiltration has attracted substantial interest because of the evidence that the occurrence of hyperfiltration may have some value in predicting the development of diabetic nephropathy3. This concept seemed plausible because of the evidence that hyperfiltration may be caused by increased glomerular capillary pressure and that intraglomerular hypertension represents a general risk factor for glomerular disease4. Despite the continuing debate about the reality behind the link between diabetic hyperfiltration and diabetic nephropathy, the issue of the causation of glomerular hyperfiltration has been intensely pursued in rodent models of diabetes. Among the proposed mechanisms responsible for diabetic hyperfiltration, relaxation of afferent arterioles in response to reduced input from tubuloglomerular feedback (TGF) has played a prominent role.The development of both type I and type II diabetes mellitus (DM) is often associated with an increase of glomerular filtration rate (GFR) , and the same phenomenon has been observed in various experimental models of DM5. Two different concepts have been advanced as to how TGF may be involved in the dysregulation of GFR in DM. One hypothesis argues that the primary process is the growth of the proximal tubule leading to enhanced water and solute reabsorption with the consequence that NaCl delivery to the macula densa decreases, the TGF-imposed vasomotor tone relaxes, and glomerular capillary pressure and GFR increase6. This “tubulo-centric” concept has been supported by substantial experimental evidence coming for the most part from experiments in rats with streptozotocin-induced type I DM6. Alternatively, it has been suggested that diabetic hyperfiltration is primary, driven by structural changes and/or by largely unknown derangements in the spectrum of vasoactive mediators, and that TGF serves as a mechanism that prevents the full extent of the effects of these abnormalities on GFR7. These two concepts are not easily reconcilable because vasorelaxation is caused by a normally functioning TGF in the first, whereas, in the second, vasorelaxation is TGF-independent and is in fact counteracted by it to the extent TGF is functional. This “glomerulo-centric” theory has found support in the finding that type I diabetic mice of the Akita strain without a functional TGF system, achieved by breeding the Ins2 mutation of the Akita mice into the TGF-less A1 adenosine receptor (A1AR) null background8, display exaggerated hyperfiltration compared to Akita mice with a presumably intact TGF9.TGF is an intrarenal regulatory system that operates at the level of the juxtaglomerular apparatus, and that translates changes in NaCl concentration at a distal tubular site, probably the macula densa, into inverse changes of glomerular capillary pressure and nephron filtration rate10. This may prevent distal NaCl levels from falling, thereby maintaining some TGF activation and preventing hyperfiltration. Even though this argument does not explain the exaggerated hyperfiltration in the Akita mice with the A1AR-/- background in which the absence of TGF makes variations of distal NaCl irrelevant, we have taken this argument as an incentive to directly assess proximal fluid reabsorption by micropuncture in Akita diabetic mice with both native and A1AR-/- backgrounds. While confirming the presence of hyperfiltration in the TGF-less diabetic mice, our data show that there are no measurable reductions in the rates of proximal fractional fluid reabsorption between WT mice and diabetic animals with or without TGF. We therefore maintain the view that, at least in this particular model of type I diabetes, TGF serves as a mechanism protecting against the development of uncontrolled hyperfiltration.In a recent extensive review of the complex issues surrounding renal function in diabetic models, it has been argued that the failure to detect a clear TGF relaxation in the Akita mouse model of diabetes might be due to excessively high plasma glucose levels and the inability of proximal tubules to enhance reabsorption sufficiently8. Genotyping of Ins2 was done by standard PCR  using AmpliTaq DNA Polymerase . The PCR product was then digested for 2 h with Fnu4HI restriction enzyme  and separated on agarose/ethidium bromide (3% [w/v]) gel to yield bands of 140 bp for WT, and 280 bp for the Ins2 mutation.Male Akita mice heterozygous for the Ins2 mutation  from Jackson Laboratories  were crossed with female C57Bl/6 WT mice in the NIH animal facility. To generate Ins2+/–/A1AR-/- double mutants, female A1AR-/- mice (C57Bl/6 background) were crossed with male F2 mice heterozygous for both the Ins2 (Akita) and A1AR mutations. All micropuncture experiments were performed in male animals Successful experiments were performed in 16 animals . Mice were housed in the NIH animal facility at a room temperature of 22°C and a 12 hour dark/12 hour light cycle. Animal experimentation was approved and carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Genotyping for A1AR was done on tail DNA using PCR as described previouslyFor micropuncture experiments, mice were anesthetized with 100 mg/kg thiobutabarbital (inactin) intraperitoneally and 100 mg/kg ketamine subcutaneously. Body temperature was maintained at 37.5°C by placing the animals on an operating table with a servo-controlled heating plate. The trachea was cannulated, and a stream of 100% oxygen was blown towards the tracheal tube throughout the experiment. The left carotid artery was catheterized with hand-drawn polyethylene tubing for continuous measurement of arterial blood pressure and blood withdrawal. A hand-drawn polyethylene catheter connected to an infusion pump was inserted into the right jugular vein for an intravenous maintenance infusion of saline at 400 µl/hr. The bladder was cannulated from a suprapubic midline incision for urine collections. Following a flank incision, the kidney was carefully dissected free of surrounding fat and placed in a lucite holder. The opening of the cup at the hilum was obstructed with fat and the kidney was covered with mineral oil.125I-iothalamate  was started 20–25 minutes before micropuncture. Nephron filtration and absorption rates were determined by free-flow micropuncture as previously described11. Following tubular identification by dye injection, proximal collections were done in the last surface segment (collection duration 2.5 min in most cases ) using oil-filled pipettes. Fluid volume was determined from column length in a 0.5 µl Drummond microcap. Samples were transferred into a counting vial and radioactivity was determined in a gamma counter . Blood samples were collected in heparinized 5 µl microcaps at the beginning and at the end of the experiment. Temporal spacing between the two blood samples was between 38 and 55 minutes. Plasma reference values for each tubular sample were obtained by linear interpolation.125I-iothalamate radioactivity was measured in duplicates using 0.5 µl samples of plasma and urine using Drummond 0.5 µl microcaps for sample transfer.To measure rates of proximal fluid reabsorption, an infusion ofAll reported statistical comparisons were made by one way ANOVA using the Bonferroni post hoc test at p<0.05 as showing significance .12. There were no significant differences between WT and diabetic mice in mean arterial blood pressure, body weight, or age. An estimate of the number of nephrons filtering at the level of measured SNGFRs (Kidney GFR/SNGFR) suggests that nephron numbers were not significantly different between the genotypes used in this study.A summary of measurements of GFR and a number of functional variables in WT (n=5), Akita (n=5), and Akita/A1AR-/- mice (n=6) during anesthesia is shown inMeasurements of SNGFR and fluid reabsorption along the proximal tubule by micropuncture confirmed the presence of hyperfiltration at the single nephron level . Mean SNUnits length of fluid column, nl total fluid volume collected, min collection time, flow flow rate (nl/min), Ucpm I125 counts per minute in total fluid volume, Bkg I125 background, column H net cpm (Ucpm-Bkg), TF tubular fluid (cpm/nl). P plasma (cpm/nl), TF/P ratio tubular fluid cpm divided by plasma cpm, SNGFR single nephron GFR (TF/P x flow rate), absorption fluid reabsorption (SNGFR - flow rate), % Del fluid delivered to collection site in % of SNGFR, % Abs fluid absorption in % of SNGFR, MAP mean arterial pressure during collection. U urine (cpm in 2 duplicates), P plasma (cpm in 2 samples in duplicates).Click here for additional data file.Units length of fluid column, nl total fluid volume collected, min collection time, flow flow rate (nl/min), Ucpm I125 counts per minute in total fluid volume, Bkg I125 background, column H net cpm (Ucpm-Bkg), TF tubular fluid (cpm/nl). P plasma (cpm/nl), TF/P ratio tubular fluid cpm divided by plasma cpm, SNGFR single nephron GFR (TF/P x flow rate), absorption fluid reabsorption (SNGFR - flow rate), % Del fluid delivered to collection site in % of SNGFR, % Abs fluid absorption in % of SNGFR, MAP mean arterial pressure during collection. U urine (cpm in 2 duplicates), P plasma (cpm in 2 samples in duplicates).Click here for additional data file.Units length of fluid column, nl total fluid volume collected, min collection time, flow flow rate (nl/min), Ucpm I125 counts per minute in total fluid volume, Bkg I125 background, column H net cpm (Ucpm-Bkg), TF tubular fluid (cpm/nl). P plasma (cpm/nl), TF/P ratio tubular fluid cpm divided by plasma cpm, SNGFR single nephron GFR (TF/P x flow rate), absorption fluid reabsorption (SNGFR - flow rate), % Del fluid delivered to collection site in % of SNGFR, % Abs fluid absorption in % of SNGFR, MAP mean arterial pressure during collection. U urine (cpm in 2 duplicates), P plasma (cpm in 2 samples in duplicates).Click here for additional data file.Units length of fluid column, nl total fluid volume collected, min collection time, flow flow rate (nl/min), Ucpm I125 counts per minute in total fluid volume, Bkg I125 background, column H net cpm (Ucpm-Bkg), TF tubular fluid (cpm/nl). P plasma (cpm/nl), TF/P ratio tubular fluid cpm divided by plasma cpm, SNGFR single nephron GFR (TF/P x flow rate), absorption fluid reabsorption (SNGFR - flow rate), % Del fluid delivered to collection site in % of SNGFR, % Abs fluid absorption in % of SNGFR, MAP mean arterial pressure during collection. U urine (cpm in 2 duplicates), P plasma (cpm in 2 samples in duplicates).Click here for additional data file.12. Similarly, the induction of diabetes by alloxan was associated with hyperfiltration in both WT and A1AR-/- mice13. The present results confirm these observations in anesthetized animals in which the GFR of Akita mice increased by 25% in the C57Bl/6 background (nonsignificant) and by 56% (p<0.05) in the A1AR-/- background. As we have argued previously, the augmented hyperfiltration cannot be mediated by TGF since the A1AR-deficiency in both mixed WT and Akita diabetic genetic backgrounds renders TGF non-functional14. We cannot exclude the possibility that A1AR-deficiency directly enhanced GFR through some unknown mechanism. However, A1AR-deficient mice have been shown previously to have normal filtration rates15 so that the GFR-raising effect would have to be linked to A1AR deficiency under diabetic conditions. The present micropuncture results corroborate the irrelevance of TGF for hyperfiltration in diabetic Akita mice in another way. Hyperfiltration at the single nephron level was seen during withdrawal of fluid at late proximal tubular sites, thereby preventing fluid from gaining access to sites beyond the proximal tubule and effectively normalizing distal fluid delivery to zero. Thus, by eliminating TGF influences, this opening of the feedback loop reveals TGF-independent effects on GFR. To the extent that TGF is functional in a given animal or condition, SNGFR measured by proximal fluid collections represents a non-steady-state that overestimates SNGFR by acute removal of the GFR-suppressing action of TGF . The relative increase in plasma glucose levels of Akita mice at a young age is about threefold based on previous measurementsorption . The inaIn summary, our results confirm at the single nephron level that GFR increases in diabetic Akita mice as a function of TGF non-functionality, consistent with the notion that TGF prevents hyperfiltration in this model of type I diabetes. Rates of absolute and fractional fluid reabsorption were found to be comparable between control and diabetic animals. While hyperfiltration is seen in both streptozotocin-induced diabetes and in the genetic diabetes of the Akita mice, the mechanisms underlying this functional abnormality may be different in the two models. The manuscript by Schnermann and associates entitled, “Nephron filtration rate and proximal tubular fluid reabsorption in the Akita mouse model of type I diabetes mellitus” addresses a critical question in the development of diabetic nephropathy. To address the critical question of the role of TGF in the glomerular hyperfiltration in diabetes nephropathy, the authors used transgenic mouse models.  Diabetic nephropathy in human patients is a complex disease and no single transgenic mouse model of diabetic nephropathy can perfectly match the human disease. Despite these limitations, Schnermann and associates defined well the problem they addressed, and used appropriate mouse models of type I diabetes mellitus and skilled techniques. In this context, their approach is well controlled and provided meaningful data vis-à-vis the problem they attempted to address. Thus, overall it is convincing to this reviewer that their conclusions are valid and acceptable to the scientific community. I do not have any major issues, and only present a few minor concerns.Although, the manuscript is well written, at some critical places the use of long sentences is not advisable to convey the meaning smoothly, especially to the not-so-experienced readers and junior scientists.It is not clear to me whether +/- data presented in the Table 1 represent SD or SEM. It should be mentioned in the Table footnote as well as under the Statistics description.Although the scattered data points show the trends, however, looking at the Akita group in Fig 1A, and Ak/A1AR-/- group in Fig 1B and 2A, it is clear that the distribution is not normal.  The Ak/A1AR-/- group has distinctive distributions as compared to the other two groups in these figure panels.  Hence, the data presented in Figures 1 & 2 will be better interpretable by including parallel box plots. The box plots also provide more insights by showing the median and quartiles.  The authors should consider this suggestion.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Dear Dr. Kishore,Thanks for your comments!You talk about model limitations in your introduction, and I wanted to confirm that we agree and are fully aware of this. One of the most intriguing aspects of the human disease is its variability in that some diabetics have hyperfiltration and some don’t, and some of those that do may get renal disease and some don’t. I am not aware that any of the experimental models mimic this critical phenotype, in part perhaps because the statistical database isn't large enough to show it. In any case, the mechanism of hyperfiltration, something that one may get out of animal studies, is much less important than its predictive value for developing renal disease, and this one can get only out of human studies. Thus, hyperfiltration in mouse models (and the mechanisms causing it) may be more of academic than clinical interest. Certainly the question of whether an increase of plasma glucose into the diabetic range increases or decreases proximal tubular fluid reabsorption is a defined question that should be answerable, and the impression that even this limited question appears to be model-dependent is somewhat disappointing.The deviations presented in Table 1 are SEMs. In regard to the figures, we thought that by showing every single measurement we leave the issue of data distribution open. Box plots don’t add much other than 25% and 75% quartiles and minimum and maximal values, and I simply doubt that there is much information in this. As to distribution, we agree that some of the data look not normally distributed, but to conclude that this is an inherent property of one strain vs. another is most likely wrong. It is much more likely that data from the 7th, or 8th, or 9th or nth mouse would fill the void and make the data more and more normally distributed. In regard to the long sentences, we know we are no Shakespeares, but the writing does not seem too bad – comparatively. The manuscript 'Nephron filtration rate and proximal tubular fluid reabsorption in Akita mouse model of type I diabetes mellitus' is a follow up to the same groups previous publication on hyperfiltration in diabetic mice model. The manuscript is very clear and easy to read despite the relatively complicated content. It addresses essential points for the nature of DM- induced hyperfiltration and the conclusion of TGF as a relative protector against DM-induced hyperfiltation is well founded. In this paper the authors look at younger mice, which is commendable as the hyperfiltration is seen as an early event in diabetes that precedes significant DM-induced renal failure. The experiments are of a high quality and the figures are generally well prepared.The current manuscript builds on previous results that firmly substantiate the used diabetic models. In this study the age of the mice are not completely comparable to the previous study. It would, however, be helpful if it was clear from the text that the relevant data is already available - that the animals in fact have high blood glucose and are seemingly slightly dehydrated. +/-; 11.5 nl/min Ins+/-A1AR-/-) – so this must apparently be new data for the 30 nl/min? – or is there some conversion of the numbers that is not perfectly clear. This point is relatively important as a fall from 15.2 nl/min  to 11.5 nl/min  in the Ak/A1AR-/- is quite substantial and does witness about some TGF in this mouse. It is unclear where the data comes from in Figure 3 (30 nl/min). The legend states that the values come from reference 9 - but the mean values for EPFR given in that publication is:  that are compared here?  Is it reasonable to make a line between the two points if they are indeed measured in separate experiments.n in the measurements of SNGFR is number of experiments and not animals.It is not explicit that the The test used to test for normal distribution of the data, which is a prerequisite for using the ANOVA test, is not stated under statistics. The values in the text are not given with the same number of decimals. The method for measuring GFR in general is not included in the method section.Minor concerns:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Dear Dr. Praetorius,      We appreciate your comments, and provide the following in response to the concerns that you have raised:   et al.Faulhaber-Walter) had an age of 14 weeks. While the age-matching between strains in the present experiments was not perfect, the mean of the ages is about 14 weeks. According to our previous data diabetic mice at this age have plasma glucose levels of between 600 and 700 mg/dl. This is an agreement with the original description of the Akita mice, in which diabetes had been shown to be of early onset with an approximate threefold increase of plasma glucose by 7 weeks of age . A manifestation of diabetes in the current experiments is the increased urine flow, which is a consequence of non-absorbed glucose as qualitatively established by dipstick, which affirms that hyperglycemia and the increase of filtered glucose must have exceeded the maximal glucose absorptive capacity.The youngest cohort of mice in our previous study . The ages of the animals used at that time were between 9.8 and 17 weeks.                It is hard to affirm Gaussian distribution if the sample size is relatively small, but all groups passed the KS test for normality. Nevertheless, subjecting the SNGFR data to a nonparametric test  showed that the probability of the null hypothesis to be correct, 0.026 suggesting that the medians between the groups are in all likelihood different.


A study of the frequency response of AFM microcantilevers in liquid media contained in a commercial fluid cell is presented. Such systems exhibit complicated dynamics which are often not well described by available theories. Their dynamic behavior has a direct effect on the use of the AFM in dynamic mode while imaging in liquid or while extracting the rheological properties of the fluid. We explore the issues related to the design of the cantilever holder/fluid cell and propose an approach for evaluating, minimizing and recognizing the ultimate limitations of commercial cantilever holders. A technique for estimating the frequency response spectrum of the fluid cell itself from experimental data is presented. This spectrum can then be used to evaluate whether or not the fluid cell is suited for the desired purpose. In a liquid environment the response of the cantilever strongly depends on the excitation technique. In the case thermal excitation , 8, the Although the thermal and magnetic driving techniques produce smoother cantilever responses, they have some drawbacks which make working with acoustic excitation desirable. Firstly, these techniques require additional hardware such as a signal conditioner, a data acquisition system, special cantilevers, and a magnetic field system making these techniques more complex and costly. Secondly, in the magnetic technique, the fluid is heated by the electromagnetic field and the magnetic coating changes the vibrational properties and bending angle of the cantilever. For these reasons, many studies have been aimed at understanding and removing the redundant peaks in the response of the cantilever to acoustic excitation.et al. [et al. [Putman et al. , who wer [et al.  observed [et al. -17. Thiset al. [et al. [Although the effects of the various design problems on the cantilever response were previously recognized, the exact relationships were not understood and improvement of the frequency response based on control of these factors has not previously been considered. Instead efforts were focused on other approaches. Tamayo et al.  mixed th [et al.  used anoet al. [et al. [Beside these practical investigations, a lot of effort has been focused on the evaluation of cantilever response theoretically. Schaffer et al.  proposed [et al.  consider [et al.  proposed [et al.  derived Also, because of the particular design of commercial fluid cells, it is impossible to apply ideal acoustic excitation to the cantilever which causes in an even more complicated frequency response. In this work, we apply some simple modifications to a widely used commercial fluid cell from Veeco   is the transverse cantilever deflection, EI is its flexural rigidity, cρ is the mass density, L is the length, A =b × h is the area of the cross section, b and h are the width and thickness of the cantilever, respectively, and hf is the hydrodynamic resistance per unit length to cantilever motion. For this equation the boundary conditions are:y(t) is the base motion. We consider w as the tip motion relative to the base, w=u-y(t), and substitute for u in the above equations. Then in the governing equation an additional term appears in the right side as an external force which is related to the inertial forces of the cantilever beam and the boundary condition at the base will change to w=0. Now by applying the Fourier transform on the above equations we find in the frequency domain:Since the internal friction of the cantilever itself is negligible compared to fluid damping, the governing equation for the deflection of the cantilever can be written in the following form:w, in the fluid. This force was determined by Stokes [fρ is the density of the surrounding fluid, b is the width of the cantilever and Γ(ω) is the hydrodynamic function that can be obtained from solving the momentum equations for the surrounding fluid.To continue, we require a general form for the hydrodynamic force. Because the amplitude of vibration of the cantilever is very small, the hydrodynamic force on each point of the cantilever can be approximated by the hydrodynamic force that would be applied on an infinitely long rigid beam that oscillates transversely with the same amplitude, y Stokes  for a ciy Stokes  modifiedi(ω)C is the complex magnitude of the ith mode, and i(x)ψ is the normalized eigenfunction of the ith vibrational mode of the undamped cantilever. The eigenfunctions are normalized in a way that i(L)ψ=1 and have the orthogonality properties:The solution of i(x)ψ and integrating over the length of the cantilever:iλ is the ith modal wavelength. herefore, given the amplitude of excitation, Y(w), we can determine the deflection of the cantilever at each point. For simplicity, we can define the following transfer function using After substituting It should be noted that the quantity measured by AFM is in fact the inclination of the cantilever. For this case, the theoretical response is simply the spatial derivative of the cantilever deflection:3.In our studies we have used four different cantilevers selected according to the requirements of each experiment. Cantilevers 1 and 2 were used in the initial experiments =1.Comparing I(ω), in the absence of fluid born excitation, can be written in the form:F(ω)T is defined as an experimentally obtained function that transfers I(ω) to the frequency response of the fluid cell itself, CT is the transfer function for the ideal damped response of the cantilever W. Note that the cantilever is excited by the function Y(w)=F(ω)T×I(ω) illustrating that ideal acoustic excitation can only be achieved if the fluid cell is designed such that F(ω)T is constant.Based on the results shown on ntilever  and togeTo further verify the linearity of the model in CT, act to amplify the dynamics of the fluid cell. When the cell is filled with air or another gas the damped cantilever response contains only sharp resonance peaks at higher frequencies and thus acts to filter out the dynamics of the cell itself.et al. [The results presented here prove that with this type of fluid cell the frequency response is dominated by the dynamics of the cell itself rather than the cantilever and that fluid borne excitation is less important than previously thought. This problem can only be solved by placing the peizoelectric actuator directly under the cantilever base as in the regular tip holders. Maali et al.  modifiedet al. [We have further demonstrated that the important design issues for fluid cells are: (1) the method of attaching the cantilever chip to the cell, (2) the location of the piezoelectric actuator and (3) the occurrence of fluid borne excitation. Of these issues, the first relates to repeatability and the second and third determine the potential for achieving ideal acoustic actuation. If the actuator is placed exactly at the base of the cantilever following the approach of Maali et al. , one can4.The frequency response of AFM microcantilevers, in a commercial fluid cell, was investigated while the cantilevers were immersed in different liquids. The dynamic characteristics of the fluid cell were determined by combining the experimental and theoretical results. It was shown that in fluid cells in which the piezoelectric element is removed from the cantilever base, ideal acoustic excitation cannot be achieved. Moreover in this case, the measured frequency response is dominated by the dynamics of the fluid cell potentially leading to significant misinterpretation of data. Contrary to previous reports, fluid borne excitation is shown to be a less significant effect.


There is an error in the funding section. The grant FONDECYT number 1070444 should be: 1100176.


In the Funding statement, a funding organization and grant providing financial support for the fourth author is incorrectly omitted.The following should be read with the Funding statement: http://www.nwo.nl) [^] (NWO Grant No. 613.000.604) of the research Council for Physical Sciences (EW).""Louis D Weise acknowledges support from the Netherlands Organization for Scientific Research (


Patients who are critically injured with imminent cardiac arrest may require immediate thoracotomy as an integral component of their initial resuscitation in the emergency department. Fluid resuscitation can be difficult owing to a shutdown state. A simple method for fluid delivery in patients requiring emergency department clamshell thoracotomy entails the insertion of a large bore venous catheter directly into the right atrium at its appendage and using it for fluid resuscitation . This me


The number of the grant from the National Natural Science Foundation of China is incorrect. The correct number is 81171506.


Several of these novel granisetron conjugates have high quantum yields and show high affinity for the human 5-HT3AR.The synthesis, photophysical and biological characterization of a small library of fluorescent 5-HT Fluorescence is a useful tool in cell biological studies, and fluorescent labeling of target proteins have enabled numerous in vivo studies of protein function.3Rs are members of the Cys-loop family of ligand-gated ion channels which also includes nACh, GABAA, and glycine receptors.3R subunits have been discovered to date (5-HT3A–5-HT3E)3R populations most likely comprise several subtypes characterized by distinct functional properties; thus human 5-HT3 signaling is more complex than originally anticipated. To date, only homomeric 5-HT3A and heteromeric 5-HT3AB receptors have been extensively characterized in heterologous systems.3A and heteromeric 5-HT3AB receptors which is consistent with their observed identical competitive pharmacologies.3R populations in native cells and tissue remains an important goal.5-HT3Rs are used in the clinic to prevent chemotherapy- and radiotherapy-induced nausea and vomiting, post-operative nausea and vomiting, for the treatment of irritable bowel syndrome, and 5-HT3R antagonists might be beneficial for the treatment of psychiatric and neurological disorders, such as anxiety, drug dependence and bulimia nervosa.Antagonists of 5-HT3R antagonist granisetron1, Ki = 1.45 nM), and identified positions on the granisetron core which were tolerant to substitution.2–5 are quite fluorescent and some of them bound with high affinity to the 5-HT3R. However, their quantum yields were poor -coumarin-3-carboxylic acid or FITC was coupled to the spacers to yield probes 36–39.Other than the N1-position on granisetron, we identified the C7-position as tolerant to substitution in our initial SAR study.rmediate . The ind16, 18 and 21) or BODIPY dyes  appended to N1 show the highest quantum yields (Φf). The quantum yield of the coumarin-containing probes 15, 17, 20, 36–38 was low in pH 7 buffer, but was substantially increased in less polar solvents  = 0.55). The binding affinities of the fluorescent granisetron probes for the human 5-HT3AR were determined by competition binding studies with [3H]granisetron. Probes 14, 16, 18, 27, 28 and 36 exhibited affinities similar to the parent compound granisetron  of transfected cells, whilst this fluorescence was absent in mock transfected cells   . In contansetron . These d3 receptor ligands. Most notably, novel granisetron conjugates 14, 16, 18, 27 and 28 have similar binding affinities for the human 5-HT3AR as measured for the parent compound 1. This is in agreement with previous studies which showed that bulky fluorophores can be appended via short aliphatic linker to the N1-position of granisetron. Moreover, these high affinity probes exhibit high quantum yields and emission maxima above 500 nm in polar media. Probes 16 and 18, in particular, show specific fluorescent labeling of the human 5-HT3AR in live cells. We are currently focusing our efforts on evaluating our probes in fluorescence polarization and flow cytometry applications.In summary, we have designed and synthesized a small library of fluorescent 5-HT


Both approaches sensitize tumours to cisplatin through the suppression of endotrophin-induced epithelial–mesenchymal transition. The beneficial effects of TZDs on cisplatin sensitivity are diminished in COL6−/− mice, whereas endotrophin+ tumours are sensitive to the TZD/cisplatin combination. Therefore, the chemosensitization obtained with TZDs is achieved through a downregulation of endotrophin. Treatment with an endotrophin neutralizing antibody in combination with cisplatin completely inhibits tumour growth of tumour allografts. Combined, our data suggest that endotrophin levels are a strong prognostic marker for the effectiveness of the combination therapy of TZDs with cisplatin, and neutralization of endotrophin activity dramatically improves the therapeutic response to combination therapy.Endotrophin is a cleavage product of collagenVIα3 (COL6A3). Here, we explore the relationship between thiazolidinediones (TZDs), endotrophin and cisplatin resistance in the context of a mammary tumour model. COL6A3 levels are increased in response to cisplatin exposure in tumours. Endotrophin, in turn, causes cisplatin resistance. The effects of endotrophin can be bypassed, either through use of COL6 null (COL6 Such ECM proteins include fibronectin, laminin, collagen I (COL1), collagen IV (COL4) and collagen VI (COL6) and these ECM components are markedly modulated in response to chemotherapy  that display a reduced EMT over the course of tumour progression, failed to show any added beneficial effects of TZDs to cisplatin. Taken together, these results suggest that the beneficial effects of TZDs on platinum-based chemotherapy are mediated through the inhibition of endotrophin in mammary tumours, and that the neutralization of endotrophin activity is a key determinant to unleash the full beneficial effects of TZDs.Recently, we identified endotrophin, a cleavage product of COL6A3 that is actively involved in mammary tumour progression through enhancing the epithelial–mesenchymal transition (EMT), fibrosis and chemokine activity, thereby recruiting stromal cells to the tumour microenvironment  mouse model or an allograft of Met-1 cancer cells (originating from MMTV-PyMT mammary tumours) that we transplanted into isogenic wild-type mice. To visualize system-wide tumour burden in vivo, we introduced an infrared-fluorescent protein (FP635) overexpressing transgene driven by the MMTV promoter (MMTV-FP635) into PyMT mice  on platinum-based chemotherapies in mammary tumour models in vivo. Nevertheless, whether and how COL6A3 directly contributes to drug responsiveness is not known.To see whether COL6 is involved in the beneficial effects of TZDs on platinum-based therapy, we determined the expression levels for COL6 in response to chemotherapy. The mRNA levels of COL6A3 in tumour tissues of PyMT mice were significantly increased in response to cisplatin treatment; this increase was dramatically suppressed by combination with TZDs . These rThe EMT process in tumour tissues is well known to contribute to an acquired drug resistance . PyMT/COL6−/− mice were more sensitive to cisplatin treatment compared to PyMT mice . We monitored this strain's cisplatin sensitivity and compared it to either PyMT or PyMT/COL6−/− mice. The mRNA levels for endotrophin in tumour tissues in PyMT/COL6−/−/endotrophin mice were about fivefold higher than those of endogenous levels in PyMT mice, whereas no changes were seen at the level of other COL6 chains (−A1 and −A2) or the remaining mature portion of full-length COL6A3 chain (COL6A3-N)  . Importasistance . Furthertitution . This pi+-tumours were more resistant to a lower dosage of cisplatin treatment  compared to control (Ctrl)-tumours (We have previously shown that MMTV-endotrophin mice bred to the PyMT mice (PyMT/endotrophin) develop more aggressive tumours compared to PyMT mice , and this increase was markedly attenuated by the combination with TZD (vs. ETP/TZD). This suggests that TZD influences not only the endotrophin expression per se, but it may also impact the downstream pathways of endotrophin. However, we cannot rule out that endotrophin-independent pathways are also contributing, or that TZD acts on host endotrophin levels in this transplantation paradigm. Defined necrotic lesion areas, as assessed by H&E stains, were significantly decreased in endotrophin+-tumours. This phenomenon was however reversed by combined treatment of cisplatin with TZD , lectin perfusion (angiogenesis) and Masson's trichrome C stain (fibrosis), respectively  on cisplatin sensitivity. Tumour pieces taken from PyMT mice were implanted into wild-type mice and treated with cisplatin alone or in combination with either TZD or 10B6 once the tumour volume reached 100 mmreatment . We alsoreatment . Nude miB6 alone . HoweverB6 alone . NotablyB6 alone . This in−/− mouse model or a TZD combination sensitizes cancer cells to cisplatin treatment. Our data presented here suggests that determining endotrophin levels in association with the EMT status is critical for predicting cisplatin response. Higher levels of endotrophin occur in advanced metastatic breast cancers  show beneficial effects in the context of cisplatin-based chemotherapy. Our previous work indicates that collagenVIα3 (COL6A3) plays an important role in cisplatin resistance. However, the detailed molecular mechanisms underlying the correlations between COL6A3 and cisplatin resistance remained elusive. The goal of this study was to elucidate the roles of endotrophin, a cleavage product of COL6A3, in cisplatin resistance and elaborate further to see if endotrophin modulates the beneficial effects of TZDs in cisplatin therapeutics in breast cancer.RESULTS:−/−) in the background of MMTV-PyMT mice, respectively. We demonstrate that endotrophin, which is mainly secreted from stromal adipocytes in the tumour microenvironment, confers a high degree of cisplatin resistance by enhancing epithelial–mesenchymal transition, fibrosis and angiogenesis. Furthermore, the powerful beneficial effects of TZDs on cisplatin sensitivity are mainly due to a marked inhibition of endotrophin-mediated activities. This suggests that TZDs directly mediate enhanced cisplatin chemosensitivity through a downregulation of endotrophin. Treatment with an endotrophin neutralizing monoclonal antibody in combination with cisplatin very effectively inhibits tumour growth of allografts of MMTV-PyMT tumours.Gain- and loss-of function studies were conducted with MMTV-endotrophin transgenic and COL6 null animals  mice were generated as previously described by Bonaldo et al  was diluted to 1 mg/ml in PBS and was sonicated briefly before injection. The PPARγ agonist rosiglitazone  was given by diet inclusion at a dose of 20 mg/kg/day BW. Anti-mouse endotrophin monoclonal antibodies  were administered by intraperitoneal injection.Formalin-fixed paraffin-embedded tissue sections were used for immunostaining. Deparaffinized tissue slides were stained with rabbit anti-mouse endotrophin, MT , E-cadherin , Vimentin  and cytokeratin . For immunofluorescence, fluorescence labeled secondary antibodies were used and counterstained with DAPI. Images were acquired using the Leica confocal microscope and analysed with ImageJ software. For immunohistochemistry, the reaction was visualized by the DAB Chromogen-A system (Dako Cytomation) and counterstained with haematoxylin. Images were acquired using the Nikon Cool Scope. TUNEL assay was according to the manufacturer's protocol . To assess functional blood vessels formation in tumour tissues, mice were injected with biotinylated tomato-lectin   and perfused lectin was visualized by a Cy3-labeled streptoavidin. H&E staining and Masson's Trichrome C staining were performed by Dr. John Shelton at the University of Texas Southwestern Medical Center. Histological analysis was performed with pathologists in the UTSW pathology core facility.Ct method using 36B4 to normalize. Primers for COL1A1, COL6A1, COL6A2 and COL6A3 were followed in previous report  using a TissueLyser  and isolated using the RNeasy kit (Qiagen). Total RNA (1 µg) was reverse transcribed with SuperScript III reverse transcriptase (Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed in the Roche Lightcycler 480. For all qRT-PCR experiments, the results were calculated using the ΔΔMammary epithelial cancer cells were isolated as described in previous report /2. Inguinal tumours was weighted to determine tumour burden. Animals were sacrificed when the tumour burden visibly affected the host or when the tumours reached the IACUC predetermined limit of 20 mm along one axis.Tumour onset was monitored twice weekly by palpation. Tumour sizes were measured with a digital caliper twice weekly and the volumes were calculated as (length × widthInfrared fluorescence expressing MMTV-PyMT mice (FP635/PyMT) were imaged by IVIS scanner  and signal intensity was analysed with Living image v.3.2 .t-test and Mann–Whitney t-test, as appropriate with GraphPad Prism v.5 software. p-value <0.05 was considered as statistical significance.All data represent mean ± SD. Data were analysed by two-way ANOVA followed by Newman–Keuls multiple comparison test or by Student's


Pancreatic ductal adenocarcinomas (PDAs) are notoriously aggressive and resistant to treatment. They distinguish themselves further by their robust fibroinflammatory, or desmoplastic, stromal reaction and degree of hypovascularity. Recent findings have revealed multiple mechanisms of stromal complicity in disease pathogenesis and resistance. In this review, we focus on altered physicomechanics as one mechanism of what we term as ‘stromal resistance’ in PDA. Extremely high interstitial fluid pressures and a dense extracellular matrix combine to limit the delivery and distribution of therapeutic agents. We discuss the genesis and consequences of altered fluid dynamics in PDA and strategies to restore them. Pancreatic ductal adenocarcinoma (PDA) is an aggressive disease of the exocrine pancreas, marked by a 5-year survival rate of <5% and a median survival of ∼6 months, making it the fourth leading cause of cancer-related deaths in the United States  tumour suppressor genes are also common  has been clinically approved, and even then provides only incremental improvement in survival . In particular, we highlight physical mechanisms of drug resistance in PDA and recent efforts to target stromal elements in order to improve delivery of small molecule therapeutics. We discuss further how studies in genetically engineered mouse (GEM) models of PDA have yielded unexpected insights compared with more traditional xenograft and allograft models and may help to explain the previous divide between preclinical promise and clinical reality.A dynamic fibroinflammatory, or desmoplastic, response is a hallmark of PDA and essentially pathognomonic for the disease. This desmoplasia evolves during disease progression and includes stromal fibroblasts, immune cells, and excessive deposition of a complex extracellular matrix (ECM) . ActivatThe delivery and distribution of small molecules across a tissue are governed by well-defined principles of solute transport involving passive diffusive and convective fluxes  which, together with the development of hypersensitivity reactions, have precluded their use as systemic agents. Chemical conjugation of recombinant human PH20 with polyethylene glycol preserves enzymatic activity and extends the half-life to >10 h. Systemic administration of this agent can deplete HA from implanted tumour xenografts in immunocompromised mice and decrease IFP  mice with established invasive PDA with intravenous injections of PEGPH20. Significant intratumoral depletion of HA and reduction of IFP occurred within 2 h and peaked at 24 h . The apparatus used to measure fluid pressure has also varied. A number of systems have been used to measure fluid and tissue pressures over the last 50 years, including the needle  are profoundly hypovascular, in contradistinction to the systems described above. Second, the vasculature is characterised by widespread collapse. Third, the tumour vessels in PDA appear to be functionally and ultrastructurally intact , to influence the fundamental strategy in treating patients with this dreaded disease.The complex cellular, molecular, and mechanical features of the desmoplastic response in PDA presents challenges to effective treatment, as well potential vulnerabilities to exploit. In creating a drug-free sanctuary, the tumour also presumably remains sensitive, at least initially, to the chemotherapies it has been shielded from rather than selected to resist. Although intrinsic mechanisms of disease resistance will no doubt emerge, breaching the sanctuary with a systemically deliverable enzyme offers the prospect of unprecedented, sustained vascular access to the PDA primary tumour bed and metastases. This will enable not only rigorous evaluation of new treatment strategies, but also exploration of distinct sequencing and schedules of various treatment modalities in the attempt to remain one step ahead of the tumour. Indeed, re-examining the long list of clinically approved agents previously thought to be ineffective in PDA would be an excellent place to start. Additional mechanisms to reduce IFP should also be explored including targeting other ECM components  or inhibiting contractile forces generated by fibroblasts. Restoring physiologic blood flow, oxygen, and nutrients to a pancreas cancer will undoubtedly alter its metabolism, and these changes may present additional therapeutic targets and also new mechanisms of resistance. However, the idea that the complex and impervious pancreas cancer ‘organ’ can be perturbed dramatically is itself encouraging. It cannot be long before this literal and figurative entry into pancreas cancer pathophysiology results in a more effective therapeutic armamentarium.


A funding organization and grant were incorrectly omitted from the Funding Statement. The following sentence should be read after the first sentence of the Funding Statement: "P.J.C. is funded by the US Public Health Service grant GM098629."


There was an error in the funding statement.The correct statement is as follows: This work was supported by research grants TBRS T13-706/11, CUHK477408, CUHK479109, and CUHK478710 from Hong Kong RGC, Strategic Investment Scheme C and Group Research Grant from Chinese University of Hong Kong, and National Science Foundation of China grant 31171100. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


A funding organization, a grant from that organization and an additional grant from FONDECYT were incorrectly omitted from the Funding Statement.The Funding Statement should read: "KSL is a Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) PhD fellow. This work was financed and supported by Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) Grant 1050433,1080075, FONDEF Grant MR10I1008 and Postdoctoral FONDECYT Grant 3130681."


A growing body of evidence indicates that digitoxin cardiac glycoside is a promising anticancer agent when used at therapeutic concentrations. Digitoxin has a prolonged half-life and a well-established clinical profile. New scientific avenues have shown that manipulating the chemical structure of the saccharide moiety of digitoxin leads to synthetic analogs with increased cytotoxic activity. However, the anticancer mechanism of digitoxin or synthetic analogs is still subject to study while concerns about digitoxin's cardiotoxicity preclude its clinical application in cancer therapeutics. This review focuses on digitoxin and its analogs, and their cytotoxicity against cancer cells. Moreover, a new perspective on the pharmacological aspects of digitoxin and its analogs is provided to emphasize new research directions for developing potent chemotherapeutic drugs. Digitalis purpurea L.) [Cardiac glycosides (CGs) are a large family of chemical compounds found in several plants and animal species . Plants urea L.) . Since turea L.) ,5.Generally, CGs share a common structural motif with a steroidal nucleus, a sugar moiety at position 3 (C3), and a lactone moiety at position 17 (C17) . CGs als+/K+-ATPase and increases intracellular sodium ions [+/K+-ATPase is a P-type pump that actively transports potassium ions inside and sodium ions outside cells in a 2:3 stoichiometry [+/K+-ATPase inhibition by CGs, the cells have to restore intracellular sodium concentration to their basal levels by stimulating the Na+/Ca2+ exchange pump to extrude sodium ions out of the cell and introduce calcium ions into the cell [It is well established that CGs inhibit Naium ions . The Na+hiometry . Such achiometry . Approprhiometry ,12. To mthe cell . This acthe cell .Bufo bufo toad secretions contains several CGs and is still being used today for managing cancerous conditions [et al. examined the cytotoxicity of CGs in rodent cancer models and found inhibition of in vitro proliferation at concentrations that are relatively toxic to humans (0.1-10 μM) [et al. compared breast cancer tissue samples from women maintained on digitalis (a CG) for cardiac conditions to tissue samples from control patients [et al. examined 127 cancer patients treated with digitalis [et al. found that those patients on digitalis treatment showed significantly lower mortality rate than the control group [A growing body of evidence suggests significant anticancer effects mediated by CGs. For instance, in the 8th century, CG plant extracts were used for treating malignant conditions . An ancinditions -16. Invenditions ,18. Shir1-10 μM) . A seriepatients . This stpatients -22. Addipatients . In 1984igitalis . While pigitalis . Twenty see Table Digitoxin is one of the few CGs that have been extensively studied and have a well-established clinical profile ,68. Digi+/K+-ATPase pump at concentrations between 0.5 - 5 μM [+/Ca2+ pump activity and subsequently induce apoptosis in cancer cells [+/K+-ATPase pump causes an accumulation of intracellular sodium, thus perturbation of cellular ion homeostasis [+/Ca2+ exchanger needs to be activated to export excess sodium to the extracellular space while importing calcium, which in turn causes an accumulation of intracellular calcium that mediates cellular events such as myocardial contractility, cytoskeleton remodeling, and apoptosis [To understand its anticancer mechanisms, initial research focused on the ability of digitoxin to inhibit Na5 - 5 μM -12,73. Ser cells -76. Figueostasis -12. For poptosis -12. Intepoptosis ,31,36,58et al. suggested that the signaling characteristics of Na+/K+-ATPase are distinct from the ion pumping activity [+/K+-ATPase signalosome is a multiple-protein signaling complex of 3 alpha (α) subunits and 2 beta (β) subunits that controls cellular activities like apoptosis [+/K+ATPase signalosome to transmit intracellular signals. Figure +/K+-ATPase at concentrations between 10-100 nM. Upon binding, digitoxin modulates the Na+/K+-ATPase protein complex activating the associated downstream signaling pathways, i.e., activation of several signaling cascades such as phospholipase C (PLC) signaling, mitogen-activated protein kinase (MAPK) signaling, phosphatidyl-inositol-3-kinase (PI3K) signaling, and Src kinase signaling.In 2003, Xie activity . It was poptosis , cell prpoptosis , cell mopoptosis , and tigpoptosis . Subsequ+/K+-ATPase have mixed functions. For example, the MAPK signaling is mainly a proliferative and pro-survival signaling that can also be complemented by a pro-apoptotic signaling capability [+/K+-ATPase signalosome is very complex. Such complexity, together with the digitoxin's narrow therapeutic window and its known cardiotoxicity, led to a slow progress in developing digitoxin as anticancer therapeutics [The signaling cascades that are stimulated upon the interaction of digitoxin with Napability -88. Src pability -97. Therapeutics ,2,18,98.et al. developed MeON-neoglycosylation, a chemoselective method for glycorandomization that employs modifying the glycosidic bond that links the saccharide moiety to the core steroidal nucleus of digitoxin [et al. and O'Doherty et al. respectively [Early on, it was suggested that a potential approach to circumvent digitoxin's cardiotoxicity is to design synthetic analogs that are either more effective or less toxic than digitoxin when used as anticancer agents ,99. Suchigitoxin ,101. Theigitoxin ,101. Figet al. compared a library of digitoxin MeON-neoglycosides and digitoxin O-glycosides in a panel of 60 different cancer cell lines [et al. used artificial O-monosaccharide analogs that they tested against a panel of 60 different cancer cell lines [et al. [The differential potency and cytotoxic selectivity of digitoxin MeON-neoglycosides and digitoxin O-glycosides have been demonstrated in various cancer cell lines. For instance, Iyer ll lines . They shll lines -39. Theyll lines -39. The  [et al. .et al. showed that the D6-MA analog was 4-5 folds more potent than digitoxin in inhibiting cell proliferation, inducing cell cycle arrest and apoptosis in NSCLC cells [Elbaz LC cells . The autLC cells . Moreove+/K+-ATPase signalosome [In order to explain the observed anticancer effect, it is postulated that digitoxin and D6-MA activate several transcriptional regulatory cascades via the Nanalosome ,102-107.+/K+-ATPase signalosome [The above suggested mechanism is supported by previous work showing that digitoxin inhibits AP-1 and NF-κB signaling through the Nanalosome ,108-112.nalosome -115. Sevnalosome ,117. Cycnalosome -123 and nalosome ,125. Thenalosome -131. Surnalosome -135. Addnalosome -139. Sevnalosome -142. Alsnalosome . Thus, bnalosome -146, a p+/K+-ATPase signalosome in a manner that significantly undermines cell viability (Figure Digitoxin and D6-MA strongly modulate cell cycle machinery through the Nay Figure . HoweverResearch findings and conclusions are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health.The authors declare that they have no competing interests.HAE drafted the original manuscript; TAS and WT coordinated and helped to draft the manuscript. YR and CZD have been involved in revising the manuscript critically for its scientific content and have given final approval of the version to be published. All the authors read and approved the final manuscript.


To overcome the limited accuracy of functional hemodynamic parameters such as stroke volume and pulse pressure variation (SVV and PPV) during spontaneous breathing, a passive leg raising (PLR) maneuver has been suggested as a reliable predictor of fluid responsiveness ,2. The a2 system. Fluid (500 ml) was given: (A) during controlled mechanical ventilation, (B) during pressure support ventilation with spontaneous breathing and (C) after extubation. The stroke volume (SV), SVV and PPV as well as the mean arterial pressure and heart rate were assessed. A PLR was performed before fluid administration at all three time points. Fluid response was defined as an increase in SV >15%. Prediction of fluid responsiveness was tested using ROC analysis.Thirty-four patients after off-pump CABG were enrolled. Measurements were performed in the ICU using a PiCCOIn 34 patients significant hemodynamic changes were observed, with 19 (55.9%), 22 (64.7%), and 13 (40.6%) responders at time points A, B and C, respectively. Prediction of fluid responsiveness is depicted in Table Prediction of a fluid response with SVV/PPV was less reliable in spontaneous breathing. PLR predicted fluid responsiveness, but was less accurate than previously reported.