Oral administration of tumour cells induces an immune hypo-responsiveness known as oral tolerance. We have previously shown that oral tolerance to a cancer is tumour antigen specific, non-cross-reactive and confers a tumour growth advantage. We investigated the utilisation of regulatory T cell (Treg) depletion on oral tolerance to a cancer and its ability to control tumour growth. Balb/C mice were gavage fed homogenised tumour tissue – JBS fibrosarcoma , or PBS as control. Growth of subcutaneous JBS tumours were measured; splenic tissue excised and flow cytometry used to quantify and compare systemic Tregs and T effector (Teff) cell populations. Prior to and/or following tumour feeding, mice were intraperitoneally administered anti-CD25, to inactivate systemic Tregs, or given isotype antibody as a control. Mice which were orally tolerised prior to subcutaneous tumour induction, displayed significantly higher systemic Treg levels (14% vs 6%) and faster tumour growth rates than controls (p<0.05). Complete regression of tumours were only seen after Treg inactivation and occurred in all groups - this was not inhibited by tumour feeding. The cure rates for Treg inactivation were 60% during tolerisation, 75% during tumour growth and 100% during inactivation for both tolerisation and tumour growth. Depletion of Tregs gave rise to an increased number of Teff cells. Treg depletion post-tolerisation and post-tumour induction led to the complete regression of all tumours on tumour bearing mice. Oral administration of tumour tissue, confers a tumour growth advantage and is accompanied by an increase in systemic Treg levels. The administration of anti-CD25 Ab decreased Treg numbers and caused an increase in Teffs. Most notably Treg cell inhibition overcame established oral tolerance with consequent tumor regression, especially relevant to foregut cancers where oral tolerance is likely to be induced by the shedding of tumour tissue into the gut. Even allowing for comparable tumour stages the prognosis for patients suffering from oesophageal and gastric cancer remains consistently and significantly poorer than for patients with distal gastrointestinal tract cancers, despite advances in diagnostic, surgical and adjuvant therapies + population, the Th3 cell +CD25+ cells in response to oral Ag administration. These Tregs were found to express CTLA-4 and foxp3 and to have a suppressive function in vitro. CD4+CD25+foxp3+ Tregs play a role in preventing the development of autoimmune disease and have a dual property as both anergic and suppressive cells. Significantly, it has been shown that removal of this population may induce anti-tumour immune activity The mechanism of tolerance to ingested Ags may be attributed to either active suppression or the induction of clonal deletion/anergy We have investigated the effects of oral tolerance to the JBS tumour on tumour growth rates and on the size of lymphocyte subpopulations in the Balb/C mouse. Specifically we examined if the induction of oral tolerance to tumours was Treg-dependent – and whether Treg inactivation could abrogate oral tolerance and inhibit tumour growth.2, in DMEM . The tissue culture media were supplemented with 10% iron-supplemented donor calf serum, 50 µg/ml gentamycin, 300 µg/ml L-glutamine, and 10 mM HEPES  monosodium salt), pH 7.4. Standard procedures for trypsinisation, centrifugation and resuspension of cells were used The JBS murine fibrosarcoma All murine experiments were approved by the animal ethics committee of University College Cork (AERR #2010/003). The study was carried out in strict accordance with the recommendations laid down by the Irish Department of Children and Health.ad libitum. Before experiments, the mice were afforded an adaptation period of 14 days. Balb/C mice of both sexes and athymic male Balb/C HsdOla:MF1-nu mice in good condition, weighing 16–22 g at 6–8 weeks of age, were included in experiments. For JBS tumour induction, 2×106 tumour cells, suspended in 200 µl DMEM, were injected subcutaneously into the flank of the mice. With this volume of inoculum and location there was little puncture site extravasation of the cell suspension and the tumour growth and shape was consistent.Mice were obtained from Harlan Laboratories . They were kept at a constant room temperature (22°C) with a natural day/night light cycle in a conventional animal colony. Standard laboratory food and water were provided 6 JBS tumour cells in Balb/C mice, tumours developed and were allowed to attain a size of 1–2 cm3, at which stage the animals were sacrificed and their tumours were removed. The tumours were homogenized in phosphate buffered saline (PBS)  using a FastPrep FP120 homogeniser . The homogenate was extensively washed by centrifugation with PBS to remove debris. When the supernatant was macroscopically clear the homogenate was resuspended in PBS to a final concentration of 0.2 g (wet weight) per ml. This was aliquoted and frozen until use. The PBS control was also aliquoted and frozen. Using an 18 Fr-steel-ball-tipped feeding needle attached to a 1 ml syringe, un-anaesthetised animals were gavage fed 200 µl of freshly thawed JBS tumour homogenate (40 mg of tumour) or PBS. Animals were fed daily for 14 days. The amount of feed and the duration of feeding corresponded to those used in previously published studies by our group and were based on a literature review, particularly in regard to studies that examined tolerance to cellular Ags 6 cells) and were monitored for tumour development daily.Following subcutaneous inoculation of 2×10v = ab2π/6 and tumour growth curves were constructed. The mice were humanely euthanised in instances where the tumour reached 1.5 cm3 in diameter. In the first experiment, mice were fed tumour Ag or PBS, and growth and survival curves were prepared. In the second experiment, systemic Tregs were either permanently inactivated (referred to as depleted) using anti-CD25 Ab , temporarily depleted during feeding only or not depleted. In the final experiment mice were fed for 14 days and subsequently randomised to receive anti-CD25 Ab or a control Ab conjugated to anti-horse radish peroxidase (HPRN), Bio-Express, USA to monitor the effect on sc tumour growth.Tumours were measured every 48 hours using a digital calliper. Tumour volume was calculated using the standard formula 4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4 with HCl) was prepared using reagents purchased from Sigma-Aldrich, Ireland. The solution was filter sterilised through a 0.2 µm filter and stored at 4°C. FACS staining buffer was prepared from Dulbecco's phosphate-buffered saline (Dulbecco's PBS) purchased from Sigma along with, bovine serum albumin (BSA), sodium azide and foetal calf serum. Spleens and tumour tissue were removed and minced separately on 70 µm nylon cell strainer . These cell suspensions were collected, washed in culture medium, pelleted at 300 g for 10 min at 4°C and were then treated with ACK erythrocyte lysing buffer for 3 min after which they were washed twice and resuspended in FACS staining buffer. Viable cell numbers were determined by a standard Trypan Blue Exclusion test. Cells were then diluted to a concentration of approximately 2–4×106 per ml and 100 µl staining buffer added per Falcon tube (Becton Dickinson). Cell suspensions and reagents were maintained at 4°C during preparation.ACK erythrocyte lysing buffer (0.15 M NHPhycoerythrin–Cy5 (PE-Cy5) conjugated antibodies against murine CD25 and CD3, fluorescein isothiocyanate (FITC)-conjugated antibodies against murine CD25, CD3, CD4 and CD8 and the PE anti-mouse foxp3 staining kit were purchased from eBioscience, Insight Biotechnology, England. PE anti murine CD4 and CD3 were purchased from Serotec, Oxford, England. Fluorochrome-conjugated isotype staining controls  were purchased from eBioscience. Unconjugated anti-murine CD16/CD32 (Fcy III/II) monoclonal Ab (Fc receptor blocking Ab) was purchased from Serotec.In order to minimise non-specific Ab binding, anti-CD16/CD32 Fc receptor blocking Ab was diluted to 0.01 µg/µl in staining buffer, 20 µl per well added and incubated at 4°C for 20 min. Fluorochrome-conjugated antibodies to cell surface markers were also diluted as per manufacturer's instructions and were added to each sample without removal of the Fc-blocking Ab. Cells were incubated in darkness at 4°C for 45 min then washed twice in 250 µl staining buffer before resuspension in 500 µl staining buffer for immediate analysis or a fixative solution of 0.5% paraformaldehyde in Dulbecco's PBS. Analysis was performed within 48 hours using a FACScaliber flow cytometer  and analysed by the accompanying CELLQuest (BD Biosciences) computer software program.As previously stated, anti-CD25 Ab (PC61) and control Ab (isotype control rat IgG-HRPN) were administered intra-peritonealy at a dose of 1 mg/kg in a total volume of 200 ul of PBS. The timing of doses depended on the experimental protocol but when two doses were to be administered they were given four days apart . This ret-test for paired values. Differences with a p value less than 0.05 were considered significant.The differences between the individual groups were tested using the two-tailed Student's We have previously shown that subcutaneous tumours have a faster growth rate in mice that were fed tumour prior to tumour induction, compared with mice that were fed either PBS or an alternative tumour (CarB or CT26) +CD25+ cells (p<0.022) following tumour administration versus PBS. Moreover there was a significant increase observed in CD25+/foxp3+ expression following gating on the CD3+CD4+ population of lymphocytes (p<0.019) (+CD4+ or CD3+CD25+ cells. Neither was there any significant increase in the CD8+CD25+ Teff sub-group populations following the tumour feeding schedule. There was no observable difference in the number or ratio of lymphocyte populations between those mice which were fed PBS and those not gavage-fed during this period (data not shown).Following 14 days of oral tumour or PBS administration, splenic lymphocyte populations were analysed. There was a significant increase in CD4p<0.019) . There w+ cells over a period of 14 days We have previously shown that our anti-CD25 Ab administration protocol facilitates 90% inactivation of CD25The resulting subcutaneous tumour growth curves showed that the previously observed tumour growth advantage following feeding was absent in mice undergoing the depletion during the tolerising schedule, with time to tumour appearance and tumour growth rate similar to PBS-fed mice, indicating the requirement for Tregs at the time of tumour feeding for the induction of oral tolerance . As seen+CD25+ Treg and CD8+CD25+ Teff numbers. Tumour feeding significantly increased systemic Treg numbers versus PBS fed groups, and this difference was also seen in anti-CD25 Ab administered mice red mice  (p<0.05)+CD25+ cells. There was an increase in the number of Teff cells that were permanently depleted and fed tumour compared with all other groups . Administration of anti-CD25 Ab immediately following tumour feeding, removed the previously observed tumour growth advantage . This waThe anti-CD25 Ab treatment did not have long term effects on Treg numbers. Mice cured of their tumours through anti-CD25 administration were studied 30 days later and had similar Treg numbers to naïve animals irrespective of whether they were from the orally tolerised or control groups .Most patients who develop cancers of the upper gastrointestinal tract die as a direct consequence of their disease. Even for those with clinically localized tumours, who are subjected to curative surgery, the five year relapse rates are usually above sixty percent +CD25+ Tregs are systemically increased in response to oral administration of whole (JBS) tumour. In addition we have shown that the induction of oral tolerance to a tumour confers a growth advantage to the cancer.We have previously shown that growth of the weakly immunogenic JBS cell line in the flank of immune competent mice led to an increase in Treg cell numbers in the tumour environment but not systemically + T cells between any of the groups suggesting that a reduction in Teffs does not occur with oral tolerance induction. It is also likely that oral tolerance and early Treg responses did not inhibit immune sensitisation to the tumour but inhibited effector function as there were no differences in the timing or completeness of tumour regression after Ab treatment between the tumour fed tolerised or the PBS control group of mice. There was a significant difference in the total number of CD4+CD25+ Tregs in mice which received anti-CD25 Ab and were tumour fed and the control saline fed groups. This was also observed in those groups which were temporarily depleted. Surprisingly however, there was no significant decrease in Tregs observed between the groups that were tumour fed and received either a dosing schedule of anti-CD25 Ab or isotype control Ab. A possible explanation for this may be due to the analysis of Treg numbers using CD4+CD25+ only at this stage of the experimental process as opposed to the more specific CD4+CD25+foxp3+. No significant decrease in Tregs may in fact involve the experimental protocol and the timing for examination of Tregs and dose of anti-CD25Ab, which was chosen based on our previous knowledge that maximum Treg depletion was within 7 days of Ab administration. The dose of Ab used in this study ensured a 90% reduction in Treg numbers in naïve mice. The overall increase in Treg numbers in response to feeding was not taken into consideration with an increased dose of Ab. Despite this, functional differences between different doses of anti-CD25 Ab were observed in the resulting growth curves, suggesting CD4+CD25+ Treg cells are involved in the T cell response that confers the tumour growth advantage following feeding. This may be due to the fact that the effect of removing or functionally inactivating Tregs with Ab depletions during the development of oral tolerance is enough to allow for partial education of the immune system and the development of anti-tumour cytotoxic responses. Interestingly, there were more cures seen in the groups that were given anti-CD25 Ab at the time of feeding only and fed tumour (75%) than in those given Ab at the time of feeding and fed PBS (60%). It may be that Ag loading with feeding and subsequent inactivation of Tregs giving rise to increased Teff numbers could have occurred and similar findings have been demonstrated elsewhere Mice whose Tregs were depleted by anti–CD25 treatment, irrespective of tumour or PBS feeding, demonstrated regression of tumours and long term cures. There were no differences in response rates between the tumour fed and control groups indicating that Treg inhibition effectively overcomes the combined oral tolerance and tumour growth influences. It is likely that the early induction of Tregs at the systemic level, achieved in this study by tumour feeding, is responsible for the tumour growth advantage observed. It is of interest that there were no differences in the numbers of CD8In the clinic, patients with GALT present with an already orally tolerised immune system, and it remains to be determined whether oral tolerance can be overcome after tumour growth has been established. In studies of tolerance, it has been shown that the adoptive transfer of Tregs into murine models can overcome immune mediated disease The results of this study suggest a mechanism of tolerance – a T-cell-mediated suppression of an antitumour immune response. The identity of the tumour Ag(s) has yet to be established; however, this study confirms and expands on observations of previous reports where feeding of tumour Ags was shown to impair antitumour cytotoxicity and thus supports the hypothesis that tumour Ags which are shed into the upper gastrointestinal tract and processed by the mucosal immune system may result in down regulation of systemic antitumour immune responses We have shown that processing of the shed tumour results in the generation of immunosuppressive Treg cell population which can attenuate the antitumour immune response and that the development of oral tolerance in this clinically based model is Treg-dependent. Moreover, we demonstrate that the abrogation of Treg cell function can be achieved with the use of anti-CD25 Ab giving rise to a systemic, non-toxic, effective anti-tumour response. Our studies show that immune modulatory effects may contribute to the significantly poorer prognosis in foregut cancers and may be overcome by immune therapy, representing a significant potential advance in the development of more effective therapies against these diseases. Anti-CD25 Treg depletion therapy has the capacity to improve prognosis and confer a survival advantage on patients with oesophageal and gastric cancers.


Varicella zoster virus (VZV) latency in sensory and autonomic neurons has remained enigmatic and difficult to study, and experimental reactivation has not yet been achieved. We have previously shown that human embryonic stem cell (hESC)-derived neurons are permissive to a productive and spreading VZV infection. We now demonstrate that hESC-derived neurons can also host a persistent non-productive infection lasting for weeks which can subsequently be reactivated by multiple experimental stimuli. Quiescent infections were established by exposing neurons to low titer cell-free VZV either by using acyclovir or by infection of axons in compartmented microfluidic chambers without acyclovir. VZV DNA and low levels of viral transcription were detectable by qPCR for up to seven weeks. Quiescently-infected human neuronal cultures were induced to undergo renewed viral gene and protein expression by growth factor removal or by inhibition of PI3-Kinase activity. Strikingly, incubation of cultures induced to reactivate at a lower temperature (34°C) resulted in enhanced VZV reactivation, resulting in spreading, productive infections. Comparison of VZV genome transcription in quiescently-infected to productively-infected neurons using RNASeq revealed preferential transcription from specific genome regions, especially the duplicated regions. These experiments establish a powerful new system for modeling the VZV latent state, and reveal a potential role for temperature in VZV reactivation and disease. in vitro or in vivo that can be experimentally reactivated. Our study establishes a system using human neurons derived from embryonic stem cells where multiple stimuli can induce reactivation from long term experimental latency. A potential role for temperature in VZV reactivation has been revealed with this system, which can now be used to study the latent/lytic switch of VZV for the first time.Most adults worldwide harbor latent VZV in their ganglia, and reactivation from it causes herpes zoster. This painful disease is frequently complicated by long-term pain, neurological sequelae, or vision loss that require improved prevention and treatment strategies. Study of VZV latency and reactivation has been severely hampered by the inability to reproduce a persistent state  The VZV latent state is established in human sensory neurons of ganglia along the entire neuraxis during primary infection and disease, chickenpox. We know little of this state and how VZV reactivates from it to cause herpes zoster. Studies exploring VZV transcription in human dorsal root ganglia (DRG) removed post-mortem by methods such as iewed in ,2] and  and in siewed in ,4]) in ) in in siewed in . While niewed in ,7]. The. Thein siewed in , that eniewed in ,10]..in situ in vitro models for latent infection that can be reactivated, there is no widely-used in vivo small animal model of latency or any in vitro system of persistent infection in which reactivation can be experimentally induced. Indeed, VZV has proven to be difficult to induce to reactivate, even from post-mortem human ganglia harboring latent VZV genomes. The strict human specificity of VZV has precluded the use of most rodents as models of latency because no animal model reproduces human disease and most rodents do not even support VZV replication. A possible exception is the guinea pig, and VZV infection of enteric neurons in vitro ). in vitro models of using cells of human origin have therefore been proposed for studying for VZV neuronal replication and persistence. These include the use of SH-SY5Y neuroblastoma derived neuron-like cells ,. We. Wein vike cells , althougke cells . We arguWe present here an extension of our previously described hESC-derived neuron model system, for which we have established conditions that lead to a prolonged, non-productive neuronal VZV infection that can be experimentally reactivated. Persistent VZV infections can be established either using acyclovir to block lytic infections, or by infection of axons without use of antiviral drugs. Persistently-infected human neuronal cultures maintain viral genomes for up to two months, with minimal transcription and undetectable translation of several VZV proteins. Importantly, renewed replication of VZV genomes and virus protein production can be initiated experimentally by interfering with growth factor-PI3 Kinase signaling cascades. We further show that stimulation combined with incubation at a reduced temperature (34°C) results in a productive, spreading infection. Comparison of the transcriptomes of quiescently vs productively-infection human neurons by RNAseq analysis reveals a preferential RNA transcription of select genomic regions during latency. In particular, the short repeated regions of the VZV genome encoding the regulatory proteins IE62 and IE63 are enriched for transcription in persistently-infected neurons. This model system should permit the elucidation of biology of VZV reactivation in detail that has not been possible until now.We have previously reported that hESC-derived neurons exposed to high MOI cell-free recombinant pOKA-derived VZV with fluorescent protein reporters of viral protein expression, results in a spreading, productive infection . In ordeWe conjecture that the combination of ACV treatment and low MOI conditions are at the threshold for generating a productive infection, resulting in ½ the wells containing GFP+ cells that overrode the ACV inhibition, while the other half were effectively prevented from expressing ORF66. No visible cytopathic effect was observed in these GFP- wells throughout the experimental period. While the establishment of these GFP-negative cultures used a low level of virus, sufficient virus per well was added that would be expected to infect at least a fraction of neurons . To addrSimultaneous digital qRT-PCR analyses of viral transcript levels for ORF63 (IE63) and ORF31 (gB) in GFP-negative wells detected low levels , whereasin situ hybridization for VZV DNA (DNA-FISH) to estimate the number of neurons harboring quiescent VZV genomes. In nuclei of productively infected neurons, the hybridization signal for viral DNA fills most of the nucleus, consistent with viral replication and nuclear modifications induced by VZV infection , while the in the second group of 5, there were transcripts at three orders of magnitude higher levels higher. Since all the longer-term neuronal cultures remained GFP-negative at the time of harvest, we hypothesize that the increase in VZV transcripts in one group of these extended cultures reflects partial release of repression of viral gene expression without progression to lytic replication and detectable GFP-ORF66 protein expression.After careful and repeated attempts using several different antibodies, we did not detect ORF63, ORF62, gI or GFP (reflecting the ORF66 protein) protein expression by immunocytochemistry in quiescently infected hESC-derived neurons. However, our cultured human stem-cell-derived neurons, like neurons in peripheral ganglia of humans and rodents, contain lipofuschin granules that are autofluorescent, and the presence of this autofluorescence may prevent unequivocal detection of very low levels of VZV proteins.It was argued above that a valid model system for the VZV latent state should be experimentally reactivatable. We therefore evaluated stimuli for their ability to drive the re-initiation of a productive infection in hESC-derived neurons quiescently infected (GFP-negative) with VZV, as measured by ORF66GFP expression. NGF signaling has been shown to be required to maintain the quiescent state of HSV in the dissociated rodent sympathetic neuron model  25], an, an25], The levels of VZV nucleic acids in wells undergoing GF withdrawal treatment were then determined and compared to those in parallel GFP-negative wells that continued to receive the three growth factors. At 2 wk post infection, GF withdrawal wells showed a modest increase in viral DNA and transcripts from ORF63 and ORF31 . CultureNGF binding to its receptor TrkA results in cellular signaling through PI3-K, and this has been shown to be important in the maintenance of the quiescent state of HSV1, since treatment with the PI3-K inhibitor LY294002 (LY) results in HSV-1 reactivation , th, thin vi Johnson . The sec Johnson ,42], lea Johnson . Since V Johnson , while win situ hybridization. Second, DNA FISH reveals small puncta of hybridization in 4–5% of the neurons, rather than large hybridization signals filling the nuclei seen in lytically VZV-infected neurons. These small foci are similar to those reported in FISH studies of quiescent HSV1  was in vivo and in vitro models for HSV1 and may apply to the other members of the herpesvirus family. It is possible that quiescently infected neurons consists of a mixed population where some are capable of responding to different reactivation stimuli, while others may not be able to support the complete reactivation process. Consequently, the low levels of reactivation seen here may reflect the number of quiescently-infected neurons able to support reactivation. Alternatively others may require different, unknown reactivation stimuli. This system may provide a platform to identify other VZV reactivation triggers.VZV in a fraction of the persistently infected neurons responded to stimuli by being reactivated in terms of expression of ORF66GFP reporter protein after prolonged periods of undetectable expression. ORF66GFP fluorescence was accompanied by increases of viral genomes and transcripts in the cultures, as well as the production of virus capable of infecting susceptible cells. However, most quiescently-infected neurons do not reactivate VZV in our cultures. About 4% of neurons contain viral genomes as detected by FISH, yet induced expression of ORF66 protein was largely restricted to few events in a culture of several thousand neurons. The lack of reactivation for some persistently infected neurons in response to experimental stimuli has also been observed for HSV1  46]. Th. Th46]. We induced reactivation using stimuli that act through the PI3K pathway, growth factor removal and PI3K inhibition. This strongly suggests that the latency/reactivation switch of VZV is controlled to some extent by the same cellular pathways that affect this switch in HSV1 . Reactivin vitro reactivation we observed was not expected. This experimental condition was evaluated because of the long standing observation that VZV replicates more efficiently at lower temperature . No. Noin vi latency . Investi latency , and/or  latency . Low lev latency  and huma latency  have beeThe VZV transcriptome in a 95% pure population of human neurons reveals that transcripts from all of the VZV genomic regions are expressed in both quiescent and productive infection, with transcription in quiescently infected neurons at a level several orders of magnitude lower. A similar finding was reported in an RNASeq study of iPSC-derived neurons infected with vOKA at low MOI . InteresS and TRS) were significantly enriched in quiescently as compared to productively-infected neurons. This suggests that the repeat regions are more transcriptionally permissive when the genome is maintained in a repressed state. Transcription of ORF63, which lies in these genomic repeat regions, has been frequently reported in models of experimental VZV latency, but our results suggest that enhanced transcription during latency may occur from a larger region than just this ORF. Interestingly, the repeat regions of HSV1 were also reported to be preferentially transcribed in a fibroblast model of persistent non-productive HSV1 infection, where the expression of IE proteins is eliminated [Strikingly, transcription of RNAs from the VZV genome mapping to the internal and terminal short repeats  preparation and concentration were performed as described previously [The H9  human embryonic stem cell line, human neonatal foreskin fibroblasts (HFF), PA6  and ARPE19  were maintained as previously described . Parent eviously .Neurons were differentiated from hESC-derived neural precursor-containing aggregates  generated by co-culturing hESC with the PA6 mouse stromal cell line as previously described . CultureAfter a minimum of 10 days of terminal differentiation of neurons into cultures that contained extensive axonal outgrowth, cells were pretreated with acyclovir  for 24 h, and then incubated with recombinant cell-free VZV expressing GFP fused to the ORF66 protein kinase. Cultures were exposed to low multiplicity of infection VZV  for two hours in the presence of ACV. After removal of virus, the neurons were maintained in the presence of ACV for 6 days. Media without ACV was then used to maintain the cultures up to 7 weeks post infection (pi), with changes twice/wk. Cultures were examined regularly over the incubation period for GFP expression microscopically. At 2, 4 and 7 weeks pi, wells that did not contain GFP+ neurons received one of two treatments to induce reactivation. Growth factor withdrawal was achieved by incubation in media lacking the three neurotrophic factors NGF, BDNF and NT3. Reactivation using PI3 kinase inhibition was performed using LY294002 hydrochloride . Where indicated in the text, LY-treated cultures were incubated at 34°C. The strategy for silent infection and reactivation is shown schematically in Microfluidic chambers with two compartments connected by microchannels  were prepared as previously described . Brieflyin situ hybridization on nuclei isolated from the neurons. A VZV DNA probe labeled with DIG was generated and hybrization detected by indirect immunofluorescence for DIG. 1684 nuclei were examined, pooled from 3 independent experiments Details of the method are in A protocol obtained from the clinical cytogenetics laboratory of Meir Hospital, Kfar Sabba, Israel was used for DNA DNA and total RNA were extracted simultaneously using Tri-Reagent (Sigma). Total RNA was reverse-transcribed using an oligo dT primer and M-MLV reverse transcriptase. Gene specific DNA probes for viral genes ORF31 and ORF 63 and human GAPDH were used for quantification with a digital PCR in duplicate samples. Copy number of viral DNA and transcripts were normalized using human GAPDH. Others have reported GAPDH expression is not affected significantly by VZV infection and is therefore a good standard for normalization of rt-PCR results . SequencCultures of human neurons were infected, and 1 week after ACV withdrawal RNA was extracted from cultures not containing any GFP+ cells or from neurons productively infected with high-MOI cell-free VZV. Total RNA  was labeled with the TruSeq Stranded Total RNA LT Sample Prep Kit (with Ribo-Zero Gold) (#RS-122-2301) and run on an Illumina HiSeq 2500 sequencer at the Crown Institute for Genomics at the Weizmann Institute, Rehovot, Israel. Analysis was performed using bioinformatics tools as described in the Live cultures were monitored with Olympus IX70 or IX81 microscopes and photographed with digital cameras. Images were enhanced using ImageJ and Paint Shop Pro software with all changes in the images  made evenly across the entire field. No features were removed or added digitally.S1 FigA) hESC- derived neuronal cultures in multiwell plates were infected with low PFU of VZV-GFP66 and incubated in the presence of acyclovir for 6 days, followed by incubation of the cultures in media without ACV. Approximately half of the wells contained some neurons that were GFP+, while the other half did not contain detectable GFP fluorescence. After 2–7 weeks further incubation, cultures received a reactivation stimulus, as detailed in the text. Neurons expressing ORF66-GFP fluorescence appeared in approximately 30% of treated wells incubated at 37°C within 2–4 days.(DOCX)Click here for additional data file.S2 FigQuantitation of genomes and transcripts was by digital PCR. Both treatments increased the levels of both viral genomes and transcripts to varying degrees at all time points tested indicating at least a partial reactivation of VZV. The data shown for the NaB experiments are averages of 3 independent experiments at each time point. The data for the combination treatments are averages of: 2 wk, 10 experiments, 4 wk, 5 experiments and 7 wk, 8 experiments.S2B Fig is a graph showing the results of treatment of cultures of hESC-derived neurons quiescently-infected with VZV with inducers of apoptosis. The treatments caused the cells to die within 4 days, but did not induce the replication of VZV genomes or transcription of VZV genes. The graph shows the average of results from 2 independent experiments.(DOCX)Click here for additional data file.S1 TableTable B provides data indicating how many initially GFP- wells were induced to contain ORF66GFP+ neurons after withdrawal of growth factors from the medium at 37°C. Table C provides data indicating how many initially GFP- wells were induced to contain ORF66GFP+ neurons after addition of a PI3K-inhibitor, LY, to the medium at 37°C.(DOCX)Click here for additional data file.S2 TableTwo biological repetitions of productive and quiescently infected neurons were analyzed and aligned to a vOka annotated genome. The number of total reads was similar for all samples. The VZV-specific reads were approximately 20–50x higher in productively infected compared to quiescently infected neurons. The RNASeq reads mapping to the invert repeat regions were done randomly and equally partitioned between the two gene copies.(DOCX)Click here for additional data file.S3 TableThe data is arranged in descending order of expression. Transcripts with significant differences in enrichment between the two groups are shaded in yellow. Note that the three most highly expressed transcripts in both types of infection are the same.(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.


Fluid resuscitation has long been considered a key intervention in the treatment of adults with severe falciparum malaria. Profound hypovolemia is common in these patients and has the potential to exacerbate the acidosis and acute kidney injury that are independent predictors of death. However, new microvascular imaging techniques have shown that disease severity correlates more strongly with obstruction of the microcirculation by parasitized erythrocytes - a process termed sequestration. Fluid loading has little effect on sequestration and increases the risk of complications, particularly pulmonary edema, a condition that can develop suddenly and unpredictably and that is frequently fatal in this population. Accordingly, even if a patient is clinically hypovolemic, if there is an adequate blood pressure and urine output, there may be little advantage in infusing intravenous fluid beyond a maintenance rate of 1 to 2 mL/kg per hour. The optimal agent for fluid resuscitation remains uncertain; significant anemia requires blood transfusion, but colloid solutions may be associated with harm and should be avoided. The preferred crystalloid is unclear, although the use of balanced solutions requires investigation. There are fewer data to guide the fluid management of severe vivax and knowlesi malaria, although a similar conservative strategy would appear prudent.The online version of this article (doi:10.1186/s13054-014-0642-6) contains supplementary material, which is available to authorized users. More than 10% of adults with severe falciparum malaria (SFM) requiring ICU admission will die from the disease ,3 and thThe implementation of guidelines that include early, aggressive fluid loading of patients with bacterial sepsis improves outcomes . In the As recently as 2010, World Health Organization (WHO) treatment guidelines observed that ‘in adults, there is a very thin dividing line between over-hydration, which may produce APO, and under-hydration contributing to shock, worsening acidosis and renal impairment’ . Against2O .,25.18,25The persisting acidosis also resulted from the fluid load’s failure to significantly improve renal function. In PRISM, no anuric patient regained a urine output with fluid loading and 75% later required renal replacement therapy (RRT). In the patients with significant renal impairment still passing urine, 50% had an increase in plasma creatinine despite 24 hours of fluid resuscitation. In VHS, renal function was as likely to deteriorate in the patients receiving a fluid bolus as in those who did not .In other series, the strongest predictor of a salutary renal response to fluid resuscitation was the renal function on admission. Only 5% of anuric patients regained a urine output with fluid loading and diuretics in a Vietnamese series . In a seIn PRISM, 74% of patients developed clinical or volumetric (EVLW of at least 10 mL/kg) evidence of APO during resuscitation. All patients were hypovolemic or euvolemic  at the point that APO developed . Fluid lThere is little pathological or radiological evidence that vasogenic edema plays a major role in the pathogenesis of cerebral malaria ,36, a prAfter 24 hours of fluid resuscitation, almost 90% of patients in the PRISM study were still hypovolemic and most had developed marked generalized peripheral edema. The patients with marked edema were more likely to develop APO and were more likely to die. Notably, in the first 24 hours, there was a median fall in the intravascular volume of the patients who later died compared with a median increase in survivors . The mecHowever, despite the documented harm of fluid resuscitation, survival in PRISM and VHS was better than expected when controlled for disease severity . This waResuscitation strategies should also be judged by the ease with which their complications can be remedied. It may be better to err on the side of under-hydrating adults with SFM as APO can occur suddenly and unpredictably and is frequently fatal even if mechanical ventilation is available. Conversely, AKI develops less abruptly and has a greater therapeutic window, allowing referral to nephrology services, where it has a better response to treatment.2/FiO2) ratio of less than 200 mm Hg died in one large series [2/FiO2 ratio of less than 100 mm Hg died in another [Despite the availability of mechanical ventilation, 89% of Indian patients with an arterial partial pressure of oxygen/fraction of inspired oxygen  were randomly assigned on admission to the hospital to receive boluses of either 20 to 40 mL/kg of 5% albumin or 0.9% saline or to receive no boluses; standard care was otherwise provided. The mortality was increased to a similar degree in the children receiving an albumin or saline bolus (relative risk of death: 1.59) compared with the control group.The fluid resuscitation of children with SFM has been explored on a larger scale than in adults, culminating in the landmark FEAST (Fluid Expansion as Supportive Therapy) study . Here, fper se, although this conclusion was somewhat hampered by limited data regarding the participants’ deaths. The mechanism for this cardiovascular collapse was unclear, although a rapid reduction in sympathetically mediated compensatory mechanisms and treatment-induced hyperchloremic metabolic acidosis were suggested. Although APO and increased intracranial pressure were suspected in some patients, neither was identified frequently [A retrospective analysis suggested that the increased mortality resulted from cardiovascular collapse rather than fluid overload equently .The hazards of fluid resuscitation are well recognized, and fluid excess has been linked to increased mortality in several critically ill adult populations -57. VariLiberal fluid resuscitation is one of the central, albeit controversial, components of the Surviving Sepsis Guidelines ,64. Alth2 is not necessarily associated with improved outcomes [Even if a patient is fluid-responsive, simply increasing DOoutcomes . If therThe WHO recommends 0.9% saline to rehydrate adults with malaria, although recent concerns about the association between the use of chloride-rich fluids and acidosis and AKI may be relevant . AlthougThere is no single fluid prescription appropriate for all adults with SFM. The disease’s protean manifestations occur in a range of patients with a variety of co-morbidities. However, in general, the available data support a conservative fluid resuscitation strategy Figure . AlthougIn the minority of adults with SFM in whom it occurs, shock remains a medical emergency, necessitating thorough assessment for concomitant bacterial infection (with initiation of broad-spectrum antibiotics) and exclusion of occult bleeding. In these patients, prompt fluid resuscitation with frequent reassessment remains the standard of care with early vasopressor support if required. Patients with a hemoglobin level of below 7 g/dL should receive fresh blood or packed cells. As anemia often evolves during treatment, clinicians should anticipate a transfusion requirement in those with more modest anemia.If patients are anuric, fluid resuscitation is unlikely to lead to a recovery of renal function and early RRT is recommended. In the setting of AKI and oliguria, small fluid boluses (5 mL/kg titrated against effect) could be trialed, although clinicians should maintain a low threshold for initiating RRT. However, if an adult with SFM has adequate blood pressure  and urine output (more than 0.5 mL/kg per hour), there appears little advantage in prescribing any fluid beyond crystalloid at a maintenance rate of 1 to 2 mL/kg per hour, with the selection of crystalloid therapy guided by plasma electrolytes.Plasmodium vivax and Plasmodium knowlesi can both cause severe disease, although sequestration is not prominent in either infection and the relative contribution of co-morbidities to clinical manifestations, particularly in vivax, is uncertain [ncertain ,74. HoweThe optimization of volume status is a fundamental aspect of critical care, and liberal fluid resuscitation is life-saving in many conditions. However, in falciparum malaria, a disease with a unique pathophysiology, it is hazardous and fails to address the microvascular obstruction seen in the disease. Adults with SFM usually have an acceptable cardiac output and blood pressure and are frequently not volume-responsive; any extra administered fluid tends to accumulate in the interstitial space, potentially exacerbating the already impaired end-organ perfusion. Liberal fluid loading in adults leads to no meaningful improvement in the acidosis and AKI and increases the incidence of APO, and in children it increases mortality in the resource-poor setting. Despite the theoretical appeal of promptly correcting hypovolemia in adults with severe malaria, clinicians caring for patients should follow the old malariologist’s adage and ‘run them dry’.


A method is proposed in order to calculate the damping effects of viscous fluids in liquid storage tanks subjected to earthquakes. The potential equation of an ideal fluid can satisfy only the boundary conditions normal to the surface of the liquid. To satisfy also the tangential interaction conditions between liquid and tank wall and tank bottom, the potential flow is superimposed by a one-dimensional shear flow. The shear flow in this boundary layer yields to a decrease of the mechanical energy of the shell-liquid-system. A damping factor is derived from the mean value of the energy dissipation in time. Depending on shell geometry and fluid viscosity, modal damping ratios are calculated for the convective component. The dynamic behavior of liquid storage tanks and of structures in general is highly influenced by the structural damping. In engineering, an ideal fluid is usually assumed in the realm of dynamic analysis of liquid storage tanks. In doing this, the potential equation of the liquid may be divided into two decoupled components: (1) the impulsive component which describes the interaction of the liquid and the shell and (2) the sloshing motion of the free liquid surface which may be accounted for by the convective component.After the modal decomposition of both components, a viscous damping is introduced to consider the dissipation of mechanical energy. The damping influences the resulting pressures as well as the amplitude of the convective fluid motion. If the response spectra method is used to calculate the dynamic response of the tank-liquid-system the spectral acceleration is determined directly by the damping ratios.The damping of the impulsive component is mainly affected by the damping of the shell, and the fluid damping may be neglected. Depending on the material of the shell, damping ratios between 2 % (steel) and 5 % (reinforced concrete) are suggested for the Serviceability Limit State . In most practical cases of tank geometry and fluid properties a damping ratio of 0.5 %  is a too optimistic assumption.The damping factor decreases with increasing fluid volume because of the descent of the surface-to-volume ratio of the fluid.The damping factor is frequency dependent; it becomes smaller for higher sloshing modes.In this paper an engineering method for the determination of viscous fluid damping in liquid storage tanks has been presented. This method was applied to the sloshing motion of the free liquid surface. Damping ratios of the convective component depending on the viscosity of the fluid and the geometry of the shell were also derived. The proportion of the damping accounted for the tank wall and the tank bottom can be distinguished and examined. Concerning the damping effects of the convective component the following conclusions can be made:It would also possible to apply the method also to the impulsive pressure component, but the impulsive component is dominated by the structural damping of the tank shell. Using normalized damping coefficients, an easy-to-use procedure for the calculation of the damping ratios is proposed and it seems to be applicable for seismic codes. It would be interesting and maybe a part of a future work to compare the predicted damping ratios with experimental results and those of more sophisticated numerical calculations.


The SC had a median thickness of 0.3 mm (0.2–0.4 mm), with no difference between tumour subtypes (p = 0.415). Additionally, no significant association between the thickness of the SC and that of the viable part of the tumour was demonstrated (p = 0.381). In conclusion our results indicate that SC thickness is relatively constant in BCC.Basal cell carcinoma (BCC) is an invasive epithelial skin tumour. The thickness of the outermost epidermal layer of the skin, the stratum corneum (SC), influences drug uptake and penetration into tumour and may thereby affect the response of BCC to topical treatment. The aim was to investigate a possible relationship between the thickness of the SC and that of the viable part of BCC. Histopathological evaluations of the corresponding SC and viable tumour thickness measurements of individual BCCs of different subtypes were explored. A total of 53 BCCs from 46 patients were studied. The median tumour thickness was 1.7 mm (0.8–3.0 mm), with a significant difference between subtypes ( The stratum corneum (SC) is the outermost epidermal layer of the skin. It consists of flattened, anucleated keratinized cells (corneocytes) enclosed in lipid bilayers including ceramides, free fatty acids, and cholesterol which together with enzymes, antimicrobial peptides, and structural proteins make a barrier function .Basal cell carcinoma (BCC) is an epithelium tumour that primarily originates in the epidermis and its appendages. It is the most common type of invasive skin cancer in the fair skinned population of the world, causing significant patient morbidity, and should be managed properly . SeveralSC thickness has been closely investigated in normal skin and has been found to vary, depending on various factors such as body site , 6. HoweA clinical estimate of BCC thickness can readily be made before selecting an appropriate therapy. Nevertheless, it is the histopathological thickness that is considered the “gold standard” measurement for prediction of treatment response. Information about the agreement between clinical and histopathological evaluations of BCC thickness is, to our knowledge, limited to the results of a single study . In thatThe main objective of the present study was to investigate a possible relationship between measurements of SC thickness and the thickness of the corresponding viable part of the BCC.The study was performed at the outpatient clinic at the Department of Dermatology, St. Olavs Hospital, Trondheim University Hospital , and approved by the regional committee for medical research ethics (REK number 4.2007.558). Patients gave written informed consent before entry into the study.Part of the study sample had been included in previous reports that compared measurements of BCC thickness from clinical investigations, punch biopsies, and excision specimens , 9. ConsThree physicians (two dermatology consultants and an experienced dermatology registrar) performed the clinical examinations of, respectively, 12, 17, and 24 tumours. A single hospital pathologist performed the histopathological assessment of all the specimens.Clinical evaluations and sampling of tumour tissue for histopathological investigations were performed on the same day. The tumour size (in mm) was clinically defined as the mean value of its measured maximum length and width. Details of study inclusions and exclusions, the particular tissue sampling procedures, and the tissue processing are given in an earlier report .BCC thickness was measured on haematoxylin, eosin, and saffron- (HES-) stained slides using an ocular micrometer (Vernier method) with a precision of 0.1 mm . The BCCp < 0.05 was considered statistically significant.All statistical calculations were performed using IBM SPSS Statistics . Visual inspection of Q-Q plots was used to examine whether data were normally distributed. The tumour size data were normally distributed and are presented as mean (SD). The tumour thickness measurements were normally distributed, whereas the SC thickness measurements were not. Therefore, all thickness data are presented as medians (quartiles). The Kruskal-Wallis test was used to compare SC thickness between subtypes. Regression analysis with tumour thickness and SC thickness was carried out to describe a possible relationship between these two parameters. A mixed linear model was used. All thickness measurement data had to be logarithmically transformed to achieve the assumption of normally distributed residuals in the models. Tumour size, location, and patient sex and age were initially included as covariates. For all tests, Measurements were taken of 53 BCCs from 46 patients. Of these tumours, 26 were located on the head/neck, 27 on the trunk, and six on the extremities. Histopathologically, 14 were superficial, 24 were nodular, and 15 were aggressive tumours. Twelve of the BCCs demonstrated crusts on histology.p < 0.001). Superficial tumours were significantly thinner than nodular and aggressive types (both p < 0.001), and there was no difference between the nodular and aggressive types (p = 0.813). The median SC thickness was 0.3 mm (0.2–0.4 mm) with no significant difference between subtypes (p = 0.415). p = 0.381). Three examples of BCCs of different subtype with different thicknesses for the viable cellular part but with similar SC thicknesses are shown in p = 0.432), tumour location (p = 0.992), patient sex (p = 0.497), and patient age (p = 0.776).The descriptive data are presented in The main finding was the no significant relationship between the thickness of the SC and that of the viable part BCC, regardless of tumour size and subtype.Knowledge of SC thickness in skin cancer is of interest in relation to PDT and may also be of interest to other topical therapies. PDT exerts its action by light activation of a photosensitizer, leading to destruction of targeted tumour cells. 5-Aminolaevulinic acid (ALA) and its ester derivate are the two most commonly used prodrugs for topical PDT of BCC . After cIn accordance with current PDT guidelines, SC and any crusts are commonly removed from the tumour surface and surrounding skin area before treatment in attempt to enhance drug permeation into tumour tissue , 17. VarWe also wanted to examine whether SC thickness is a factor that may affect clinical assessment of tumour thickness. In an earlier aforementioned study, the results demonstrated that the clinicians overestimated the thickness of thin tumours and underestimated the thickness of thick tumours compared to histology . The his in situ and invasive squamous cell carcinomas has been studied previously, and no significant difference in SC thickness between the different histological diagnoses was shown [The thickness of the SC in nonmelanoma skin cancers such asas shown . Our resas shown . Resultsas shown , 6, 20. as shown , 22.The study results indicate that the thickness of SC in BCC is fairly constant independent of the viable tumour thickness and across different tumour sizes and subtypes.


Background: Evidence suggests that Src homologous protein phosphotyrosyl phosphatase 2 (SHP2) mutations promote cancer development in several solid tumours. In this study, we focused on the in vivo and in vitro effects of an SHP2 mutation on the breast cancer phenotype to determine whether this mutation is correlated with a malignant phenotype.Methods: Mutant PTPN11 cDNA (D61G) was transduced into MDA-MB231 and MCF-7 cells. The effects of the D61G mutation on tumourigenesis and malignant behaviours, such as cell adhesion, proliferation, migration and invasion, were examined. Potential underlying molecular mechanisms, i.e., activation of the Gab1-Ras-Erk axis, were also examined.Results:In vitro experiments revealed that tumour adhesion, proliferation, migration and invasion were significantly increased in the SHP2 D61G mutant groups. Consistently, in vivo experiments also showed that the tumour sizes and weights were increased significantly in the SHP2 D61G-MB231 group (p < 0.001) in association with tumour metastasis. Mechanistically, the PTPN11 mutation resulted in activation of the Ras-ErK pathway. The binding between Gab1 and mutant SHP2 was significantly increased.Conclusion: Mutant SHP2 significantly promotes tumour migration and invasion at least partially through activation of the Gab1-Ras-Erk axis. This finding could have direct implications for breast cancer therapy. Breast cancer is the most common malignant tumour affecting women worldwide. It is the second leading cause of cancer-caused mortality in women in the US. The most important factors contributing to this high mortality rate are metastasis and recurrence. Studies have reported the overexpression of Src homologous protein phosphotyrosyl phosphatase 2 (SHP2SHP2) in breast cancer tissues. However, the role of the SHP2 signalling pathway in the malignant progression of breast cancer remains unknown.Xenopus gastrulation and to cause tail truncation . Transwell chambers with polycarbonate filters of 8-μm porosity (BD Biosciences) were used in this study. In invasion assay, the chambers were pre-coated with 100 μl of 5 mg/ml Matrigel  and loaded into 24-well culture plates. In migration assay, the chambers were loaded into 24-well culture plates without Matrigel pre-coating. The transwell insert consisted of upper and lower chambers separated by a membrane with an 8-μm pore size. MDA-MB231 cells  with a 1.2% agarose underlay. The number of colonies was counted after 18 days. For focus formation assay, cells were reseeded and cultured for 4 weeks in DMEM with 10% FBS. The cells were then fixed with formalin and stained with 0.1% crystal violet.Anchorage-independent growth, which is a characteristic of 3 mutant or control MCF-7 cells were re-suspended in DMEM medium and were then seeded on top of the coated chamber slides. The cells were cultured for 30 min, and then the media was replaced with 150 μl of 5% Matrigel. After 15 min of incubation, 500 μl DMEM was added. The top layer of mediumwas changed every four days.To investigate the effect of tumour angiogenesis, we utilised a three-dimensional (3D) model. Eight-well chamber slides were pre-coated with 150 μl Matrigel. A total of 2.5×106) were diluted with PBS to a total volume of 0.1 ml and injected into the mid-dorsa of BALB/c nude mice (4-6 weeks old). The animals were inspected weekly for tumour development. Growing tumours were measured using Vernier callipers, and tumour volumes were calculated using the formula volume = length × width2 × 10. Based on the tumour volume data, the mice were sacrificed, and the tumours were isolated at 50 days post-injection. Upon tumour removal, tumour volumes were calculated using the equation tumour volume = (length × width2)/2. The tumour samples were fixed in 10% neutral buffered formalin for further analysis.Tumour cells  were cut and stained with haematoxylin and eosin (H&E).All experiments were repeated a minimum of 3 times. The data are shown as the mean value ± standard deviation. The groups were compared using one-way ANOVA, followed by Pearson's coefficient analysis of bivariate correlation. A p-value of below 0.05 was considered significant.


Reason for Corrigendum:There was a mistake in the grant number in acknowledgment section as published. The correct version appears below. The authors apologize for the mistake. This error does not change the scientific conclusions of the article in any way.This paper was supported by the Polish National Center of Science grant UMO-2011/03/B/NZ9/01577 to AOS. PP is funded by grant no. NRF-2013R1A2A2A01016282 from the Korean National Research Foundation of the Republic of Korea.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.This paper was supported by the Polish National Center of Science grant UMO-2011/03/B/NZ9/01577 to AOS. PP is funded by grant no. NRF-2013R1A2A2A01016282 from the Korean National Research Foundation of the Republic of Korea.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There are some missing funding sources for this article. Please refer to the correct funding statement below:The studies in this manuscript were funded by Exelixis Inc.; PO1 National Institutes of Health grant (PO1CA085859); Pacific Northwest Prostate Cancer SPORE grant (P50CA097186); Prostate Cancer Foundation; Richard M. Lucas Foundation; and University of Washington Bridge funding. Two of the co-authors (DTA and FS) are employees of Exelixis. They were involved in study design, data analyses, decision to publish and preparation of the manuscript.


Nature by Momo Bentires-Alj’s laboratory demonstrate that withdrawal of anti-CCL2 treatment accelerates lung metastasis and death in mice. The study highlights the need to consider longer term consequences of therapeutic intervention of metastatic disease, especially with regard to transient interference with the tumour microenvironment.Neutralisation of macrophage chemoattractant C-C chemokine ligand 2 (CCL2) has shown reduced metastasis and enhanced survival in numerous experimental models of tumorigenesis. However, important new findings reported in  The association between macrophage accumulation in primary tumours and poor prognosis is well established, and it is widely acknowledged that macrophages can contribute to metastatic onset . In breaBonapace and colleagues’ article demonstrates mammary tumour secretion of CCL2, enabling recruitment of inflammatory monocytes to the primary tumour and the metastatic site . The autBonapace and colleagues’ study identifies the surprising consequences of termination of an immunotherapy that has previously shown promise both in the laboratory and in clinical application ,6-8. TheThe immune system must have the ability to detect foreign antigen and respond in an appropriate and timely manner, but must also be adequately desensitized to avoid unnecessary response to self. The majority of studies into CCL2 signalling describe its tumorigenic features, but CCL2 is also known to provide protection, for example, by recruiting anti-tumorigenic T cell subsets to the tumour microenvironment . TherefoTo determine this hypothesis, further clarification is required to understand the extent to which monocytes are sequestered in the bone marrow during CCL2 neutralisation. It is difficult to interpret the distribution of these cells without appropriate data outlining their localisation and abundance in a resting mouse. This is exacerbated by differing patterns in monocyte distribution between experimental cell lines. Future studies must address bone marrow output during treatment to fully decipher whether monocytes are sequestered here. It is acknowledged that cancer patients often have augmented production of immature cells ; therefoCCL2 neutralisation has shown positive outcomes in several murine models of mammary, prostate and lung cancers ,12-14. D


Salmonella induces the up-regulation of connexin 43 (Cx43), a ubiquitous protein that forms GJs. This up-regulation allows the transfer of antigens between tumour cells and dendritic cells (DC) licensing them to induce an efficient anti-tumour response in a mouse model of melanoma .Most cancer cells down-regulate gap junctions (GJ) resulting in loss of communication with their surrounding microenvironment. We have previously shown that infection of mouse tumour cell lines with Herein, we tested the idea that through the formation of intercellular communication channels in tumour cells, human autologous dendritic cells could be loaded with tumour antigens, preprocessed by cancer cells, and could be used to generate a cancer vaccine.Salmonella Ty21a (Vivotif) to induce up-regulation of Cx43 and formation of GJ channels. HLA matched DCs were differentiated in vitro by peripheral blood purified monocytes from healthy donors. Expression of surface molecules, cytokine release and cell proliferation was determined by cytofluorimetry. Cytotoxicity was determined either by Delfia cell cytotoxicity assay (PerkinElmer) and by a cytofluorimetry based CD107 assay.Human melanoma cell lines were infected with a vaccine strain of Vivotif-infected tumor cells established GJs in human melanoma cell lines. We show that tumour derived antigens could transit from 'donor' melanoma cells' cytoplasm to DC's cytoplasm through GJs, generate a tumour-specific CTL response and reactivate tumour infiltrating lymphocyte (TIL) in vitro. The transfer of tumor antigens was GJ-dependent because it was abolished in the presence of a GJ-specific inhibitor. In vitro generated tumour-specific human CTLs lysed also non-donor melanoma cell lines indicating that DCs were loaded with tumour-associated antigens shared among melanoma cell lines.Moreover, in vitro generated CTLs were melanoma specific because they were unable to lyse HLA matched colorectal adenocarcinoma cell lines.We exploited an antimicrobial response present in tumour cells to activate cytotoxic CD8 T cells specific for tumour-peptides, through a new pathway of antigen loading of human dendritic cells via intercellular communication channels. This unique and novel approach can be applied across a wide range of tumour cell types and can be used clinically as therapeutic vaccine alone or in combination with gold standard treatments.


The administration of an appropriate volume of intravenous fluids, while avoiding fluid overload, is a major challenge in the pediatric intensive care unit. Despite our efforts, fluid overload is a very common clinical observation in critically ill children, in particular in those with pediatric acute respiratory distress syndrome (PARDS). Patients with ARDS have widespread damage of the alveolar–capillary barrier, potentially making them vulnerable to fluid overload with the development of pulmonary edema leading to prolonged course of disease. Indeed, studies in adults with ARDS have shown that an increased cumulative fluid balance is associated with adverse outcome. However, age-related differences in the development and consequences of fluid overload in ARDS may exist due to disparities in immunologic response and body water distribution. This systematic review summarizes the current literature on fluid imbalance and management in PARDS, with special emphasis on potential differences with adult patients. It discusses the adverse effects associated with fluid overload and the corresponding possible pathophysiological mechanisms of its development. Our intent is to provide an incentive to develop age-specific fluid management protocols to improve PARDS outcomes. Of note, due to the relative novelty of the PALICC definition, the articles included in this review still employ the Berlin or older AECC definition of ARDS.Pediatric acute respiratory distress syndrome (PARDS) is one of the most challenging disease entities in the pediatric intensive care unit (PICU). PARDS was recently defined by the Pediatric Acute Lung Injury Consensus Conference (PALICC) group as acute-onset hypoxic respiratory failure with new infiltrate(s) on chest radiography not fully explained by cardiac failure or fluid overload . This dePatients with PARDS form a heterogeneous population within the PICU. This is due to the large age distribution, as PARDS affects infants to adolescents, and the variety of underlying triggers, such as pneumonia, sepsis, trauma, and aspiration. Importantly, differences in the pathophysiology between PARDS and adult ARDS are likely. This is best illustrated by the lower incidence  and lower mortality (18–35% versus 27–45%) of ARDS in children as compared with adults , as to cAppropriate fluid management has become an important non-pulmonary treatment in the current “best practice” care of adult patients with ARDS. To prevent aggravation of lung edema, it is now generally recommended to avoid positive cumulative fluid balance and treat fluid overload after initial hemodynamic stabilization. Several studies in adult ARDS patients have studied the effect of a restrictive fluid management protocol . Most imIn this manuscript, we review the literature on the adverse effects of fluid overload with emphasis on potential physiological differences in fluid homeostasis between children and adults. In addition, we summarize the current evidence for a restrictive fluid management protocol specifically in PARDS by a systematic literature review.In adults, multiple studies in patients with ARDS have indicated higher cumulative fluid balance is associated with worse disease outcome. There is an evident inverse correlation between number of ventilator-free days (VFDs) and cumulative fluid balance –23. In tAs mentioned above, there are distinct differences in incidence and outcome between adult ARDS and PARDS. As fluid overload is an important and much prevalent aspect of this disease entity, we deem it likely that physiological differences between adults and children in fluid homeostasis influence the progression and outcome of PARDS. Differences in fluid homeostasis exist between children and adults. In humans, aging is associated with a decrease in total body water (TBW) . The TBWIn critically ill children, including those with PARDS, it is likely, although yet hypothetical, that age-dependent changes in ICW and ECW as described above alter their susceptibility for adverse effects in end organs during fluid overload. For example, young children have a large brain to intracranial volume ratio as compared with adults, possibly making them relatively vulnerable to encephalopathy as a result of brain edema by excess of water. This is for example illustrated by the higher susceptibility for cerebral edema accompanying diabetic ketoacidosis in children when compared with adults , 29. In Fluid overload in children with PARDS, and more broadly in all critically ill patients, may have an adverse effect on clinical outcome by leading to interstitial edema resulting in impaired oxygenation and perfusion of several tissues in the human body. Increased hydrostatic pressure as a consequence of intravascular fluid challenge is a major mechanism of this increase in extravascular water content. However, the development of interstitial edema by fluid overload is not solely due to heightened hydrostatic pressure as explained by the Starling forces . In addiFluid overload can have deleterious consequences for many end organs. In patients with PARDS, the lungs appear particularly affected and the aggravation of lung edema is therefore considered a key mechanism of adverse outcome by fluid overload. Extravascular accumulation of protein-rich edema in the lungs is a hallmark of PARDS and occurs by disruption of the alveolar–capillary barrier. The principal proinflammatory, cell death, and proteolytic and coagulation pathways underlying this increased lung permeability have been reviewed elsewhere , 44. As Other tissues, especially encapsulated organs such as the kidneys and liver, may be specifically prone for adverse effects of fluid overload by lower perfusion pressures. This could be explained by the capsule acting as an unyielding shield leading to a type of compartment syndrome –51. For In summary, fluid overload may be associated with negative outcome in critically ill children including those with PARDS, although important differences between children and adults in susceptibility and pathophysiology may exist. In order to address the current evidence that advocates or would support a restrictive fluid management specifically in PARDS, we performed a systematic review as described below. An overview of the search, screening and selection process of the studies can be found in Figure post hoc analysis. Table All studies included in this systematic review were part of a prospective or retrospective observational cohort or p < 0.001 and 1.08, 95% CI 1.01–1.15, p = 0.02, respectively) (p = 0.11). In the post hoc analysis by Willson et al. the increased cumulative fluid balance (as averaged per meter squared of body surface) was associated with in-hospital mortality on days 1–7 (p < 0.001) in children with ALI/ARDS  was identified according to the AECC criteria of ARDS. With regard to fluid data, they showed that non-survivors of AHRF had higher median fluid balances, although this was statistically not significant (p = 0.079). The mortality of patients with a daily fluid balance ≤10 ml/kg/day was lower than the group with a balance of >10 ml/kg/day (p = 0.049). There were no fluid data presented of only the ARDS subgroup. The last included study by Randolph et al. on the relation between weaning duration and fluid balance in mechanically ventilated children in the PICU held no data on mortality  in both bivariate and multivariate regression  . Valentictively) . A trendALI/ARDS . Hu et aALI/ARDS . A subgrortality .p = 0.01, 0.01, and 0.05 for days 2, 3, and 4, respectively) (p < 0.001) . Duration of weaning showed a non-significant relation with higher cumulative fluid balances at ERT ; however, it was  significant when predicted by the cumulative fluid balance values at extubation . An important note to be taken into account when comparing the study of Randolph et al. to the others is that ARDS was not defined explicitly or analyzed separately.The other primary and important outcome measure was the number of VFDs measured at day 28 after admission or duration of ventilation. VFDs were used in three of the five included studies –61 and ictively) . Likewis< 0.001) . Their pp = 0.011)  .early fluid overload is of main interest, as this seems to negatively affect clinical outcome. Late fluid overload may have a more modest effect on outcome  . In multsize of the effect cannot be readily compared due to the nature of the retrospective studies in children and lack of prospective randomized trials.Although fluid restriction could be one of the strategies to prevent or overcome fluid overload, it is at the same time one of the main impeding factors in achieving energy requirements . Accomplearly fluid overload was found to be of main influence. However, definite evidence by randomizing liberal versus restrictive fluid treatment in PARDS is lacking. This systematic review indicates the overall effect of fluid overload on outcomes is rather similar to the effect seen in adults; however, the size of effect cannot be compared at this time. Given the accumulating evidence for differences in (patho)physiology and outcome between children with PARDS and adults with ARDS, as well as known differences in fluid homeostasis related to aging, further study of the effects of specific tailor made fluid maintenance strategies in PARDS is highly warranted. In particular, the place for early diuretic and renal replacement therapy in order to limit fluid overload need to be explored. Further studies are needed to determine whether development of specific age-related fluid management protocols, taking into account all aspects of PARDS, is essential for optimal treatment of this patient group.Pediatric acute respiratory distress syndrome is a complex disease entity in need of multimodal therapy, of which fluid treatment is a major component. The current literature on fluid balance in PARDS is sparse but demonstrates that fluid overload is associated with worsening clinical outcome, such as fewer VFDs and worse oxygenation. Varying evidence was found on the relation with mortality and increasing fluid balance. In particular, SI designed the review, conceptualized the systematic literature search, conducted the eligibility screening, assessed the quality of selected studies, extracted data from the selected studies, and drafted the initial manuscript. RA conceptualized the design of the review and critically revised the manuscript. JD conceptualized and performed the systematic literature search and critically revised the manuscript. JL conceptualized the design of the review and critically revised the manuscript. RB designed the review, conducted the eligibility screening, assessed the quality of selected studies, supervised the data extraction, collaborated in drafting the initial manuscript and critically revised the manuscript. JW designed the review, supervised the literature search and data collection, and critically revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The present study deals with the flow and heat transfer analysis of two immiscible fluids in an inclined channel embedded in a porous medium. The channel is divided in two phases such that a third grade fluid occupies the phase I and a viscous fluid occupies the phase II. Both viscous and third grade fluids are electrically conducting. A constant magnetic field is imposed perpendicular to the channel walls. The mathematical model is developed by using Darcy's and modified Darcy's laws for viscous and third grade fluids respectively. The transformed ordinary differential equations are solved numerically using a shooting method. The obtained results are presented graphically and influence of emerging parameters is discussed in detail. Fluid flow nested with porous medium is a common phenomena in nature such as transport of water in living plants, trees and fertilizers or wastes in soil. In the industry, the flow through porous medium has acquired much attention in the view of its promising applications in several technological processes for example, filtration, catalysis, chromatography, petroleum exploration and recovery, cooling of electronic equipment.The practical applications of the non-Newtonian fluids in engineering and industry have captivated a number of investigators during the past few years. Some of the important applications are aerodynamic heating, electrostatic precipitation, petroleum industry, synthetic fibres, manufacture of intumescent paints for fire safety applications and paper production. Several models have been proposed to describe the physical behaviour and properties of non-Newtonian fluids. These models exhibit a non linear relationship between the stress and rate of strain. Amongst them, the fluid of third grade, which form a subclass of the fluids of differential type , is consFrom the industrial and engineering point of view, the study of two-phase flow is of considerable practical interest. Counter current liquid-liquid flow is an example of two phase flow in an inclined channel and has applications in process industry. Ullmann et al.  investigAll the studies in the above paragraph pertain to the study of two immiscible fluids in a channel in the absence of porous medium. However, the flow problem through a porous medium channel finds many applications in geothermal and geophysical applications. These applications include underground disposal of nuclear wastes, spreading of chemical pollutants in water-saturated soil, migration of moisture in fibrous insulation, structure reaction and gas assisted injection molding, die filling processes, clean-up of refineries and extraction of geothermal energy. Nakayama et al.  made an The aim of the present investigation is to study the problem of laminar flow of incompressible and electrically conducting immiscible fluids in an inclined channel through a porous medium. In the present study we have analyzed a two-phase flow down an inclined plane by considering one of the fluid as a third grade non-Newtonian fluid. The third grade fluid exhibits the shear thinning characteristics, therefore the proposed results modify the previous analysis when both the fluids are taken to be Newtonian. The channel is filled with two immiscible fluids namely viscous fluid and the third grade fluid. A transverse magnetic field is applied perpendicular to the flow of the fluid. Section 2 contains the details of the developed mathematical model. The detail of the numerical method used is presented in section 3. Numerical results and their discussion are presented in section 4. Section 5 contains some concluding remarks.x–and z–direction making an inclination ϕ with the horizontal. The geometry of the problem is presented in Y ≥h1 and—h2≤Y ≥0 are denoted as phase I and phase II, respectively. The phase I is filled with an electrically conducting third grade fluid of density p1, viscosity μ1, electrical conductivity σ1 and thermal conductivity K1 where as the phase II is occupied with an electrically conducting viscous fluid of density p2, viscosity μ2, electrical conductivity σ2 and thermal conductivity K2. A uniform magnetic field of strength B is applied perpendicular to the flow field and the flow is saturated in porous medium. The fluid properties of both the fluids are taken as constant and the flow of both phases is driven by constant pressure gradient (-∂p /∂x). The walls are maintained at constant temperature Tw1 and Tw2 at y = h1 and y = h1 respectively. Under these assumptions, the governing equations for the flow phenomena in the vector form are given asi = 1,2 respectively describe the phases I and II, d./ dt is the material derivative, J is the electric current density, Ri are the Darcy’s resistances, Li are the velocity gradients, cp is the specific heat, g is acceleration due to gravity, pi are densities, βi are the coefficient of thermal expansions, Ti are the temperatures, Ki re the thermal conductivities, τ1 and τ2 are the Cauchy stress tensors for third grade fluid and viscous fluid respectively defined asp is pressure, I is identity tensor, α1,α2 and β3 are the material constants. The Rivlin Ericksen tensors are defined ask* and φ are the permeability and porosity of the porous medium respectively.We consider a steady, incompressible, laminar and fully developed flow between two infinite parallel plates extending in ui  are the x- component of the velocities.For the present problem the velocity field is given byUsing Equations –10) in  in 10) iFor Phase IFor Phase IIGr is the Grashof number, Ec is the Eckert number, Pr is the Prandtl number, Re is the Reynolds number, M is the Hartmann number, β is the third grade parameter, λ is the porosity parameter, P is the nondimensional pressure gradient and With reference to the With the above nondimensional quantities, our problem takes the following formPhase IPhase IIEc) because in most of the practical problems the Eckert number is very small and is of order 10−5 (see [The asterisks have been dropped for simplicity and ε <<1 (= Pr 0−5 (see ).The velocity, temperature and interface boundary conditions (15) and (16) in nondimensional form areβ,λ,m,s,h and φ In order to solve the Equations  by  by β,λ,mp1, -(21), (25), (26), (27) and (28) with the initial conditions (22)-(23), (29), (30), (31) and (32) respectively. The values of parameters P1, P2, s1 and s2 are modified by using a suitable zero finding algorithm. In the present case we have used Newton’s method so that–6 is achieved.The above process is repeated until an accuracy of 10β, the porosity parameter λ, ratios of viscosities m, electrical conductivities s, heights of the fluids h and the inclination of channel ϕ. For physical illustrations of the numerical results Figs. Gr = 5, Re = 5, Pr = 5, k = 1, n = 105, M = 2 are fixed in this study.The solution of the problem in terms of velocity and temperature distributions is obtained numerically by employing a shooting method with Runge-Kutta algorithm for various values of the parameters for example the third grade parameter u(y). is shown for several values of the third grade parameter β in β. The cause for this change is the shear thickening effects which are increased by increasing value of the third grade parameter β. Moreover, it is also observed that the decrement in the fluid velocity in the upper phase is larger as compared to the fluid velocity in the lower phase due the presence of third grade fluid. λ on the fluid velocity u(y). The effect of the porosity parameter is to suppress the fluid velocity u(y). in both the phases because of the dampening effects of the Darcy's resistance. m on the fluid velocity u(y). From this figure, it is evident that the effects of ratio of viscosities m is quite interesting and the flow field is larger in the lower phase due to the smaller value of the viscosity of the fluid compared to the fluid in the upper phase. ϕ on the fluid velocity u(y). It is noticed from this figure that an increase in the angle leads to a decrease in the velocity of the fluid and the flow field is larger in the case of third grade fluid. s on the fluid velocity u(y). One can see from this figure that an increase in the ratio of the electrical conductivities reduces the magnitude of fluid velocity. It is further noted that the flow field is larger in the case of third grade fluid. u(y) for several values of the ratio of heights of the fluid h From this figure, it is evident as we increase the ratio of heights of the fluid (or channel) the magnitude of the fluid velocity decreases. It is noteworthy that in all the above figures, the present results are compared with the existing results of Malashetty et al. [β = 0) and clear medium (λ = 0), and found them to be in good agreement.The variation in the fluid velocity y et al.  for the θ(y) for different values of the third grade fluid parameter β It is noted from this figure, that an increase in third grade parameter β results in the reduction of fluids’ temperature. The variations in the temperature distribution θ(y) for several values of the magnetic parameter M are presented in M Furthermore, the change in the temperature distribution is more visible in the case of magnetic field as compared with third grade parameter β. m on the temperature profile θ(y). It can be examined that as the ratio of viscosities increases the temperature of the fluid decreases. Moreover, the effect of the ratio of viscosities m on temperature θ(y) is same as that of its effect on the velocity field. h on the temperature field θ(y). It is noticed from this figure that the magnitude of the temperature is larger in the upper phase due to its small height compared to the lower phase. From this figure, it is also evident that the effect of the ratio of heights h on temperature profile is same as its effect on the velocity field.u(y) decreases with the increasing value of the third grade parameter β, the porosity parameter λ and the magnetic parameter M.The fluid velocity θ(y) decreases with the increasing value of the third grade parameter β, the porosity parameter λ and the magnetic parameter M throughout the channel.The temperature distribution The velocity field in upper phase is large due to the presence of third grade fluid compared to the lower phase.u(y) and temperature field θ(y) are increased as we increase the values of h and m.Both the fluid velocity In this work, a study of mixed convective hydromagnetic flow of two immiscible fluids namely the third grade fluid and the viscous fluid in an inclined channel through a porous medium has been carried out. The obtained dimensionless ordinary differential equations governing the problem are solved numerically. The effects of numerous physical parameters on the fluid velocity and temperature distributions were discussed with the help of several graphs. Following conclusions have been made from this investigation which are as follows: