VHL locus. Although this did not support tumour association with VHL disease, it was not possible to look for a loss-of-function mutation. The association of primary microcystic adenoma of the sinonasal cavity with VHL disease remains speculative. These lesions are benign but are likely to require regular surveillance. Such tumours may require repeated surgical excision. Primary clear cell microcystic adenoma of the sinonasal cavity is rare. It has previously been described only as a VHL-associated tumour. Von Hippel-Lindau (VHL) syndrome is an inherited cancer syndrome characterised by an elevated risk of neoplasia including clear cell renal cell carcinoma (ccRCC), haemangioblastoma, and phaeochromocytoma. We describe the second reported case of a primary clear cell microcystic adenoma of the sinonasal cavity. The 39-year-old patient with VHL syndrome had previously undergone resection and ablation of ccRCC. He presented with epistaxis. Imaging demonstrated a mass in the ethmoid sinus. Initial clinical suspicion was of metastatic ccRCC. However, tumour morphology and immunoprofile were distinct from the previous ccRCC and supported a diagnosis of primary microcystic adenoma. Analysis of DNA extracted from sinonasal tumour tissue did not show loss of the wild-type allele at the VHL tumour-suppressor gene with loss of the wild-type allele  in a VHL disease-associated organ system leading to tumour formation [Von Hippel-Lindau (VHL) disease is a cancer syndrome characterised by an increased risk of multiple tumour types occurring across different organ systems. These include haemangioblastomas within the central nervous system and characteristic visceral lesions including clear cell renal cell carcinoma (ccRCC) and phaeochromocytoma . Microcyormation , 3.Sinonasal tumours are rare , 5. Comm VHL mutation in 2004. He had previously undergone excision of cerebellar haemangioblastoma in 2003. He had also undergone bilateral nephron sparing surgery for ccRCC  in 2005 and renal radiofrequency ablation in both 2008 and 2012. CT imaging demonstrated an ethmoid mass measuring 35 × 34 × 44 mm. The patient underwent endoscopic transsphenoidal resection of the lesion. Staging investigations were negative for metastatic disease.A 39-year-old man with molecularly confirmed VHL disease presented with epistaxis in 2012. Molecular analysis had confirmed a germlineTwo years later, in 2014, the patient developed symptoms of nasal obstruction. Sinonasal tumour recurrence was suspected. Imaging demonstrated limited progression in the nasal cavity. Excision biopsy was performed. Histology appeared consistent with the initial presenting sinonasal tumour. Excision appeared complete, with no evidence of residual tumour. Subsequently, symptoms recurred. Therapeutic excision was performed in 2015 and again in 2016. To date, the patient's VHL disease remains stable with no evidence of new lesions or metastatic disease.The patient remained under the management of specialist multidisciplinary (MDT) team which coordinated regular imaging surveillance and clinical review. The MDT recommended regular magnetic resonance imaging (MRI) surveillance with localised resection as applicable. More extensive surgery or adjuvant therapy was not felt to be indicated.Slides of the sinonasal tumour resected in 2012 and the patient's ccRCC resected in 2005 were collated to allow comparison. Immunohistochemical and special stains for CD10, RCC, CK7, CK20, epithelial marker AE1/3, vimentin, EMA, Ki67, ssms1, sma, alpha-inhibin, p63, thyroglobulin, NSE, s100, GFAP, and PAS were performed according to standard automated protocols .The sinonasal tumour demonstrated tubulocystic morphology with a brush border, glycogen-rich cells, and low grade nuclei without conspicuous mitoses Figures . The renThe sinonasal tumour was positive for CK7, epithelial marker AE1/3, vimentin, NSE, and EMA, with patchy CK20 staining, but negative for RCC and CD10. Secretions were PAS-positive and Ki67 demonstrated a low proliferation index. Immunohistochemistry revealed renal tumour positivity for RCC, CD10, and EMA and negativity for CK7 and CK20 Figures . VHL confirmed by germline DNA analysis in 2004. DNA extracted from the sinonasal tumour resected in 2012 underwent molecular analysis in 2015 for loss of heterozygosity (LOH) at the VHL locus.The patient was known to have a constitutional deletion of exon 1 of VHL (Genbank accession number NM_000551.3) was carried out by direct sequencing analysis of sinonasal FFPE-extracted tumour DNA. PCR products were generated using a PCR reaction with a 25 μl volume and a 60°C annealing temperature using exon primers; 1F-tccgacccgcggatccc, 1R-tcagaccgtgctatcgtcc, 2F-gacgaggtttcaccacgtta, 2R-tcaagtggtctatcctgtact, 3F-tcgttccttgtactgagacc, and 3R-gtaccatcaaaagctgagatg. For the exon 1 fragment 5 μl of Q solution was added to the PCR reaction . Products were sequenced using the Big-Dye® Terminator v1.1 Cycle Sequencing Kit standard protocol  and separated on an ABI 3130xl Genetic Analyzer . Data was analysed using Mutation Surveyor (version 3.1) software . Dosage analysis was attempted using the Multiplex Ligation-dependent Probe Amplification (MLPA) method [Point mutation analysis of the three coding exons of) method , but thiAnalysis of tumour DNA demonstrated no causative mutations; however, the tumour was heterozygous for an intron 1 polymorphism (c.341-50G>A), reducing the likelihood of LOH.Primary tumours of the nasal cavity are rare , 5. MetaIn the case we present here the initial clinical suspicion was of metastatic ccRCC. Histologically, the clear tumour cells, cystic morphology, and positive staining for AE1/3  and vimeIn the previously reported case of a primary clear cell microcystic adenoma of the nasal sinus, Xu et al. describe microcystic tumour morphology, clear glycogen-rich cells without nuclear atypia, EMA and cytokeratin positivity, and negativity for CD10 . These f VHL LOH [ VHL locus. The presence of an intron 1 polymorphism makes LOH unlikely. Therefore, molecular analysis did not support tumour association with VHL disease. However, it was not possible to test for a loss-of-function tumour mutation. It is also feasible that heterogeneity of the tumour means that this result is not truly representative of LOH status.Xu et al. demonstrated sinonasal microcystic adenoma VHL LOH . This co VHL LOH . In the In the World Health Organisation (WHO) classification of head and neck tumours, adenomatous lesions of the sinonasal cavity are of salivary gland origin . HoweverThe sinonasal microcystic adenoma described by Xu et al. was surgically resected due to local progression. However, the clinical course of such tumours is unknown . In the In conclusion, the association of primary microcystic adenoma of the sinonasal cavity with VHL disease is possible but not proven. Such lesions are rare and may provide a considerable diagnostic challenge. Importantly, these lesions are considered essentially benign. However, they may have potential for rapid growth and local recurrence. This suggests that in such cases regular surveillance is merited and that repeated surgical resection may be required.


Glioblastoma is a highly aggressive and malignant type of cancer that is apoptosis resistant and difficult to cure by conventional cancer therapies. In this regard, an oncolytic adenovirus that selectively targets the tumour tissue and induces tumour cell lysis is a promising treatment option. We designed and constructed a hypoxia-responsive and cancer-specific modified human telomerase reverse transcriptase (H5CmTERT) promoter to drive replication of an oncolytic adenovirus (H5CmTERT-Ad). To enhance the anti-tumour efficacy of H5CmTERT-Ad against malignant glioblastoma, we also generated an H5CmTERT-Ad expressing secretable trimeric tumour necrosis factor-related apoptosis-inducing ligand (H5CmTERT-Ad/TRAIL). H5CmTERT promoter-regulated oncolytic adenoviruses showed cancer-specific and superior cell-killing effect in contrast to a cognate control oncolytic adenovirus replicating under the control of the endogenous adenovirus promoter. The cancer cell-killing effects of H5CmTERT-Ad and H5CmTERT-Ad/TRAIL were markedly higher during hypoxia than normoxia owing to hypoxia responsiveness of the promoter. H5CmTERT-Ad/TRAIL showed more potent anti-tumour efficacy than H5CmTERT-Ad did in a xenograft model of TRAIL-resistant subcutaneous and orthotopic glioblastoma through superior induction of apoptosis and more extensive virus distribution in the tumour tissue. Altogether, our findings show that H5CmTERT-Ad/TRAIL can promote dispersion of an oncolytic adenovirus through robust induction of apoptosis in a highly TRAIL-resistant glioblastoma. Therefore, new therapeutic strategies for the effective treatment of glioblastoma are needed. Cancer gene therapy, which delivers a therapeutic gene into tumour cells, is a promising alternative to standard care3. To date, adenoviruses have been the most frequently utilized gene delivery vectors in clinical trials of gene therapy4. A cancer-specific and replication-competent adenovirus, i.e. an oncolytic adenovirus, is particularly promising for cancer gene therapy because the oncolytic adenovirus possesses intrinsic anti-tumour activity through replication-mediated lysis of cancer cells5. After cell lysis, amplified oncolytic adenovirus progenies are released and invade neighbouring cancer cells through secondary infection, ultimately resulting in a potent oncolytic effect due to lateral spread of the virus throughout the solid tumour8.Glioblastoma is the most aggressive, invasive, and common form of human glioma. Despite decades of intensive research and advances in conventional anti-cancer modalities, patients with glioblastoma have the mean life expectancy of only 14.6 months12. Prostate-specific antigen (PSA)-, α-fetoprotein (AFP)-, carcinoembryonic antigen (CEA)-, or other cancer type-specific promoters induce efficient therapeutic gene expression in a cancer cell-specific manner12. However, these cancer type-specific promoters can target only a single type of cancer expressing tumour antigens14. In stark contrast, the promoter of human telomerase reverse transcriptase (hTERT) is active in most types of tumours, namely, 90% of tumours strongly express telomerase, while its activity in healthy cells is minimal16. hTERT is the primary determinant of telomerase activity, and its activity can be increased by c-Myc- and Sp1-mediated regulation17. A modified hTERT promoter containing additional c-Myc- and Sp1-binding sites (mTERT) has been shown to induce stronger transcriptional activity than wild-type hTERT promoter can in tumour cells18, revealing that oncolytic adenoviruses replicating under the control of the mTERT promoter are good candidates for the treatment of cancers from various tissues of origin.Utilization of a cancer cell-specific promoter has shown great potential for expressing exogenous genes in tumour tissues2 level of 1.3%20. Hypoxia is a critical hurdle for the development of a successful treatment regimen against glioblastoma because hypoxia is known to make tumour cells more resistant to radio- and chemotherapy23. Furthermore, overexpression of hypoxia inducible factor (HIF)-1α during hypoxia promotes tumour growth24. Moreover, hypoxia attenuates viral replication of an oncolytic adenovirus in tumour tissue26. Therefore, a novel strategy is needed to deal with the hypoxic tumour microenvironment within a solid tumour and to improve the anti-tumour efficacy of oncolytic adenoviruses.The tumour microenvironment is known to be hypoxic, having a median O2. Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a strong therapeutic candidate for the treatment of glioblastoma because TRAIL can potently induce cancer-specific apoptosis27. Induction of TRAIL protein-mediated apoptosis in various types of tumour cells causes effective inhibition of tumour growth without substantial toxicity in various preclinical models33. The full-length TRAIL structural analysis by crystallography enabled the development of a secretable and trimeric form of TRAIL  containing a secretion signal, a trimerization domain, and an apoptosis-inducing domain of the TRAIL protein36. Secreted proteins, which circulate throughout the body, often have better access to target tissues than non-secreted proteins do, and this attribute is particularly important for therapeutic efficacy mediated by ligand molecules like TRAIL37.Malignant gliomas are known to be highly resistant to apoptosis, which is the main mechanism behind clinical benefits of radiation and chemotherapyIn the present study, we generated a stTRAIL-expressing oncolytic adenovirus replicating under the control of a hypoxia-responsive and cancer-specific H5CmTERT promoter (H5CmTERT-Ad/TRAIL) to achieve robust and selective cancer cell-killing effect for the treatment of glioblastoma. We demonstrate that H5CmTERT-Ad/TRAIL can efficiently replicate and spread within a brain tumour, thus showing potent anti-tumour efficacy mediated by robust induction of apoptosis. Thus, H5CmTERT-Ad/TRAIL is a promising therapeutic option for the treatment of aggressive glioma.39. Furthermore, several oncogenic transcription factors, such as c-Myc and HIF-1α, have been reported to transactivate the hTERT promoter17. We previously reported that an oncolytic adenovirus replicating under the control of mTERT containing an additional c-Myc-binding site and Sp1 sites has a potent and cancer-specific anti-tumour effect that is superior to that mediated by a wild-type hTERT promoter-driven oncolytic adenovirus18. In view of these findings, we inserted five additional c-Myc-binding sites upstream of the mTERT promoter, thus generating a 5CmTERT promoter to enhance the promoter transcriptional activity in cancer cells. 5CmTERT-Ad showed significantly higher cancer cell-killing efficacy than mTERT promoter-driven oncolytic adenovirus (mTERT-Ad) did, indicating that insertion of five additional c-Myc-binding sites greatly augmented transcriptional activity of the mTERT promoter  upstream of the 5CmTERT promoter, thereby generating a hypoxia-responsive and cancer-specific H5CmTERT promoter. This promoter was utilized to drive replication of the H5CmTERT-Ad with the aim of enhancing the activity of the control oncolytic adenovirus (Rb7Δ19) replicating under the control of the endogenous adenovirus early region 1A (E1A) promoter, possesses mutated retinoblastoma (Rb) binding site in the adenovirus E1A gene, and deletion to E1B 19 kDa region under hypoxic conditions .Another critical hurdle for an oncolytic adenovirus is the hypoxic tumour microenvironment, which attenuates viral replication and the spread of the oncolytic adenovirusons Fig. . To prefP < 0.05, P < 0.01, or P < 0.001 versus Rb7Δ19 for U87MG, P < 0.01 or P < 0.001 versus Rb7Δ19 for U251N, respectively) had a dose-dependent cancer cell-killing effect that was superior to that of the Rb7Δ19 in glioblastoma cells. Furthermore, H5CmTERT-Ad/TRAIL elicited a more potent cancer cell-killing effect than H5CmTERT-Ad did during normoxia  and hypoxia . These data indicated that stTRAIL expression can augment the potency of the oncolytic adenovirus. At the highest viral dose examined, H5CmTERT-Ad/TRAIL elicited a 4.2- or 2.0-fold stronger cancer cell-killing effect than H5CmTERT-Ad did in glioblastoma cells  during normoxia. Moreover, HRE-containing oncolytic adenoviruses (H5CmTERT-Ad and H5CmTERT-Ad/TRAIL) showed markedly higher cell killing efficacy during hypoxia than normoxia , indicating that HRE inserted upstream of a cancer-specific promoter can give hypoxia responsiveness to (and enhance the potency of) an oncolytic adenovirus. Of note, H5CmTERT-Ad and H5CmTERT-Ad/TRAIL were not cytotoxic toward normal cells , thus demonstrating good cancer specificity of the H5CmTERT-based oncolytic adenoviruses. These results also illustrate that the expression of stTRAIL did not harm normal cells, confirming that the stTRAIL-mediated cell-killing effect is indeed cancer specific. Taken together, these results indicate that the stTRAIL-expressing oncolytic adenovirus replicating under the control of the hypoxia-responsive and chimeric mTERT promoter had a potent and cancer-specific cytocidal effect under normoxic and hypoxic conditions.To assess the cancer cell-killing effect and cancer specificity of the oncolytic adenoviruses during normoxia and hypoxia, a 3--2,5-diphenyltetrazolium bromide (MTT) assay was performed on glioblastoma cell lines (U87MG and U251N) and normal cell lines (BJ and SVG-P12) after infection of these cells with one of oncolytic adenoviruses at varying multiplicity of infection (MOI). As shown in Fig. P < 0.05 or P < 0.001). Altogether, these results indicate that oncolytic-adenovirus-mediated expression of stTRAIL can enhance the anti-cancer effect of an oncolytic adenovirus via induction of apoptosis.To test whether the increased cell-killing effect of the stTRAIL-expressing oncolytic adenovirus was mediated by the induction of apoptosis, a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay was performed. Camptothecin (CPT), a well-established chemical inducer of apoptosis, was used as a positive control for analysis of apoptotic cancer cell death. In both U87MG and U251N glioblastoma cells, H5CmTERT-Ad/TRAIL treatment yielded a markedly greater number of TUNEL-positive cells than Rb7Δ19 or H5CmTERT-Ad treatment did during normoxia, showing a 4.0- or 2.3-fold (U87MG) and 4.7- and 1.6-fold (U251N) higher percentage of apoptotic cells, respectively Fig. . Moreove40 . It should be noted that H5CmTERT-Ad/TRAIL elicited 1.5-fold higher tumour growth inhibition than H5CmTERT-Ad did, indicating that oncolytic adenovirus-mediated stTRAIL expression in tumour tissue can enhance the anti-tumour efficacy of an oncolytic adenovirus (P < 0.05).To evaluate the anti-tumour effect of H5CmTERT-Ad/TRAIL, the growth of established U87MG xenograft tumours was monitored following intratumoural injection of the phosphate-buffered saline (PBS), H5CmTERT-Ad, or H5CmTERT-Ad/TRAIL. As shown in Fig. rd day after final treatment and subjected to histological, immunohistochemical, and immunofluorescent analyses. As shown in Fig. P < 0.01 versus H5CmTERT-Ad), indicating that oncolytic-adenovirus-mediated expression of stTRAIL can enhance anti-tumour efficacy of an adenovirus by promoting apoptotic cell death , suggesting that stTRAIL-induced apoptosis may facilitate secondary infection of neighbouring tumour cells and enhance the viral distribution of the oncolytic adenovirus in tumour tissue.To evaluate the mechanism underlying the potent anti-tumour effect of H5CmTERT-Ad/TRAIL, U87MG xenograft tumours were harvested on the 3ath Fig. . Moreovein vivo preclinical models for testing new drugs and therapies because of their close resemblance of clinical cancer41. To determine whether H5CmTERT-Ad/TRAIL can suppress the growth of glioblastoma in its natural niche, orthotopic glioblastoma xenografts were established by stereotaxically injecting U87MG glioblastoma cells into the left forebrain of athymic nude mice. On the 7th day after cell injection, the mice were intracranially injected with PBS, H5CmTERT-Ad, or H5CmTERT-Ad/TRAIL. As shown in Fig. th day after the cell injection. In contrast, mice treated with an oncolytic adenovirus (H5CmTERT-Ad or H5CmTERT-Ad/TRAIL) had a significantly reduced tumour area in comparison with those treated with PBS; H5CmTERT-Ad and H5CmTERT-Ad/TRAIL caused 61.7% and 89.8% suppression, respectively . It should be noted that H5CmTERT-Ad-treated tumour tissues showed a 73.4% smaller tumour region than did those treated with H5CmTERT-Ad (P < 0.05), indicating that stTRAIL expression by H5CmTERT-Ad/TRAIL can enhance the anti-tumour effect of the oncolytic adenovirus against orthotopic glioblastoma. Importantly, H5CmTERT-Ad/TRAIL induced potent tumour growth inhibition up to 21 days post cell injection in U87MG/Fluc orthotopic glioblastoma tumour model (P < 0.01 for H5CmTERT-Ad/TRAIL versus PBS) . Importantly, H5CmTERT-Ad/TRAIL-treated mice showed 7.5-fold higher tumour growth inhibition than H5CmTERT-Ad-traeted mice at 21 days post cell injection. Furthermore, 33.3% of H5CmTERT-Ad/TRAIL-treated mice were viable at 35 days post cell injection, whereas only 16.7% of H5CmTERT-Ad-treated mice were viable in the same time period. Together, these results suggest that H5CmTERT promoter-regulated oncolytic Ad can effectively suppress the growth of aggressive orthotopic glioblastoma tumours through expression of TRAIL gene.The orthotopic tumour models are reliable BS) Fig. , showingP < 0.05 and P < 0.01, respectively). Additionally, immunohistochemical analysis of serially sectioned U87MG/Fluc-harboring brain tissues was performed. In line with our results from Fig. To verify the mechanism underlying the improved therapeutic efficacy of H5CmTERT-Ad/TRAIL, U87MG orthotopic tumours were harvested 17 days after cell injection and then evaluated by immunohistochemical analysis. A large amount of stTRAIL was observed in the tumour tissues treated with H5CmTERT-Ad/TRAIL Fig. . In addi42. However, the results of initial gene therapy trials in glioblastoma have been disappointing due to limited therapeutic gene transfer efficiency and insufficient cancer specificity43.Malignant gliomas remain devastating primary brain tumours and such patients have a poor survival rate. Glioblastoma is characterized by uncontrolled cellular proliferation, propensity for recurrence, and resistance to apoptosis; these properties severely restrict the efficacy of standard treatments. In this regard, cancer gene therapy has emerged as a promising approach to treatment of glioblastoma44 and (2) insertion of HREs augmented the promoter activity under hypoxic conditions47. Of note, HRE-mediated enhancement of transcriptional activity of the H5CmTERT promoter did not affect cancer selectivity of the oncolytic adenovirus because both versions of H5CmTERT-Ad killed normal cells negligibly under normoxic and hypoxic conditions. These data are in good agreement with another report showing that the addition of HREs upstream of a cancer-specific promoter does not impair this promoter’s cancer specificity under hypoxic conditions48.To overcome these limitations, we generated a chimeric hTERT promoter (H5CmTERT) containing multiple copies of HREs and several additional c-Myc-binding sites to drive the replication of an oncolytic adenovirus Fig. . Under n49, mammalian target of rapamycin (mTOR)50, or nuclear factor (NF)-κB53 signalling pathway55. It is noteworthy that the strong transcriptional activity of the H5CmTERT promoter enabled H5CmTERT-Ad/TRAIL to express stTRAIL abundantly and led to effective induction of apoptosis in TRAIL-resistant U87MG31 and TRAIL-sensitive U251N57 glioblastoma cells /Aktlls Figs  and 3, slls Figs . These fin vivo. Of note, the magnitude of apoptosis and viral distribution also showed a strong positive correlation in the glioblastoma xenograft model: H5CmTERT-Ad/TRAIL (with a higher magnitude of apoptosis) also yielded more extensive viral distribution within tumour tissue than H5CmTERT-Ad did. This phenomenon can be mediated by two distinct mechanisms: (1) stTRAIL expression increases formation of apoptotic bodies, and these apoptotic bodies transfer viral progenies to neighbouring cancer cells61, and (2) extensive apoptotic cell death can generate a void and decrease the interstitial pressure within a solid tumour, ultimately facilitating lateral spread of the virus through enhanced diffusion62. In good agreement with these reports, H5CmTERT-Ad/TRAIL-mediated apoptotic cell death was associated with large numbers of virions in cellular debris, suggesting that the oncolytic adenovirus may be transferred to neighbouring tumour cells by apoptotic bodies , glioblastoma cell lines (U87MG and U251N), and normal cell lines (BJ and SVG-P12) were purchased from the American Type Culture Collection . All the cell lines were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% of foetal bovine serum , 2 mM L-glutamine (Gibco BRL), and 100 IU/mL penicillin-streptomycin (Gibco BRL). All the cell lines were maintained at 37 °C in a humidified atmosphere containing 5% of COSix-week-old athymic nude mice  were maintained in a laminar air-flow cabinet with specific pathogen-free conditions. All facilities were approved by the Association for Assessment and Accreditation of Laboratory Animal Care. All animal studies were performed according to the institutionally approved protocols of Hanyang University.E1A gene and deletion in the E1B 19-kDa region (pDE1sp1B/Rb7Δ19)69 as a template plasmid. First, the H5CmTERT promoter was inserted into pDE1sp1B/Rb7Δ19, resulting in the pDE1sp1B/H5CmTERT/Rb7Δ19 adenovirus E1 shuttle vector. For homologous recombination with the linearized adenovirus dE1, the XmnI-treated pΔE1sp1B/H5CmTERT/Rb7Δ19 adenovirus E1 shuttle vector was co-transfected into Escherichia coli BJ5183, generating pH5CmTERT-Ad. The proper recombinant adenovirus DNA was digested with PacI and transfected into HEK293 cells to generate an H5CmTERT-Ad. The propagation, purification, and titration of all the adenoviruses used in this study were performed as previously described70. The number of viral particles (VPs) was calculated from optical density measurement at 260 nm (OD260), where absorbance of 1.0 (OD260 = 1) was equivalent to 1.1 × 1012 VP/mL. Purified viruses were stored at −80 °C until use.To generate an oncolytic adenovirus, a vector based on human adenovirus serotype 5, adenovirus E1A gene was placed under the control of the H5CmTERT promoter. We used the adenovirus E1 shuttle vector containing mutated a Rb-binding site in the adenovirus stTRAIL gene excised from pGT2SEC(CV)ILZTRAIL37 was first subcloned into the adenovirus E3 shuttle vector using BglII, creating the pSP72-E3/TRAIL E3 shuttle vector. The newly constructed pSP72-E3/TRAIL E3 shuttle vector was then linearized with XmnI digestion, and H5CmTERT-Ad was linearized with SpeI digestion. The linearized pSP72-E3/TRAIL E3 shuttle vector was then co-transfected into E. coli BJ5183 along with the SpeI-digested pH5CmTERT-Ad for homologous recombination, resulting in the pH5CmTERT-Ad/TRAIL adenovirus vector. Lastly, HEK293A cells were transfected with viral DNA to generate H5CmTERT-Ad/TRAIL as described above.To generate an H5CmTERT promoter-regulated oncolytic adenovirus expressing stTRAIL in the adenovirus E3 region, we first constructed a pSP72-E3 adenovirus E3 shuttle vector expressing stTRAIL. The 8) were plated in a 100-mm3 culture dish and then infected with one of oncolytic adenoviruses  at MOI of 0.5. To remove pre-existing human TRAIL from the medium, cells in each well were washed three times with 3 mL of the serum-free medium 30 h prior to harvesting and were incubated in a serum-free medium for additional 6 h. The cells were then further incubated for 24 h in the DMEM medium containing 5% of FBS. TRAIL expression was determined using a human TRAIL ELISA kit .U87MG cells  in PBS (2 mg/mL) was added into each well. After 4 h incubation at 37 °C, the supernatant was discarded and the precipitate was dissolved in 200 μL of dimethyl sulfoxide (DMSO). The plates were then analysed on a microplate reader at 540 nm. Absorbance recorded in uninfected cells was assumed to represent 100% cell viability.U87MG and U251N cells were plated on coverslips and treated with 1 μM CPT, Rb7Δ19, H5CmTERT-Ad, or H5CmTERT-Ad/TRAIL at MOI of 1.0. After 48 h, the TUNEL assay was carried out according to the manufacturer’s protocol . The cells were counted  in five randomly selected high-power fields .3 dish were infected with Rb7Δ19, H5CmTERT-Ad, or H5CmTERT-Ad/TRAIL at MOI 1.0. After 36 h, the cells were harvested and prepared as described elsewhere71.U87MG cells grown to 80% confluence in a 60-mm7 cells into the abdomen of 6-week-old male athymic nude mice (Orientbio) . When tumours reached an average volume of 100–150 mm3, the mice were intratumourally injected with PBS, H5CmTERT-Ad, or H5CmTERT-Ad/TRAIL (5 × 109 VPs) on days 1, 3, and 5. The first day of treatment was designated as day 1. Tumour growth was monitored every other day by measuring the length (L) and width (W) of each tumour with a caliper, and tumour volume was calculated according to the formula: tumour volume = 0.523 × L × W2.To assess the anti-tumour effect of oncolytic adenoviruses, U87MG xenografts were subcutaneously established by injecting 10Tumour tissues were collected from mice 3 days after the final treatment. Tumour tissues were fixed in 4% formalin and embedded in paraffin. Representative tissue slides were stained with H & E. Tumour slides were also stained with a mouse anti-TRAIL antibody (Ab) (BD PharMingen) to assess the expression of stTRAIL in tumour tissue. After overnight incubation with primary Abs at 4 °C, tumour slides were processed with an ABC-peroxidase kit . To assess viral replication in tumour tissues by immunofluorescence analysis, the slides were stained with a rabbit anti-E1A IgG Ab  and then incubated with an Alexa Fluor 568-conjugated anti-mouse IgG (secondary) Ab . Nuclear staining with 4′,6-diamidino-2-phenylindole  was also performed. The induction of apoptosis (TUNEL-positive spots) in tumour tissue was assessed by means of a TUNEL assay kit (Merck). All slides were counterstained with Mayer’s hematoxylin. The expression levels of stTRAIL and TUNEL-positive cells (apoptotic cells) were semi-quantitatively analysed using the MetaMorph image analysis software . E1A expression was semi-quantitatively analysed in the ImageJ software .4 cells/4 μL in Hank’s buffered saline solution (HBSS)] into the left forebrain at the following coordinates: 2.0 mm lateral and 1 mm anterior to bregma at 3.0 mm depth from the skull surface under anaesthesia (n = 4). Seven days after the tumour cell injection, the mice were intracranially injected with PBS, H5CmTERT-Ad, or H5CmTERT-Ad/TRAIL (5 × 109 VPs) at a constant pumping rate of 0.8 μL/min. At 17 days after the cell injection, all the animals were euthanised. To evaluate tumour by histology and immunohistochemistry, the brains were removed, placed in a 4% paraformaldehyde solution, embedded in paraffin and cut into 5-μm sections . Representative slices were stained with H & E and then examined by light microscopy. The same paraffin slides were also processed for detection of TRAIL- and TUNEL-positive spots as described above. Tumour slides were also stained with a rabbit anti-E1A IgG Ab (Santa Cruz Biotechnology) and then treated with an ABC-peroxidase kit  to assess viral replication in tumour tissue. The expression levels of TRAIL-, E1A-, and TUNEL-positive cells were semi-quantitatively analysed by means of the MetaMorph image analysis software .Six-week-old male athymic nude mice (Orientbio) were subjected to a stereotaxic implant of U87MG cells [7 × 109 VPs) at a constant pumping rate of 0.8 μL/min. Tumour growth was measured every 7 days after the first treatment by bioluminescence imaging using the IVIS imaging system . In vivo bioluminescence signals were calculated as the sum of the both prone and supine acquisitions for each mouse after background subtraction of total flux [photons/s (p/s)] from a total body region of interest. Cancer progression was monitored until a predetermined endpoint based on overall health. Survival curve was then plotted against time after treatment. To evaluate tumour tissue by histology and immunohistochemistry, the brains were removed, placed in a 4% paraformaldehyde solution, embedded in paraffin and cut into 5-μm sections (Wax-it) at 10 days after the cell injection. Representative slices were stained with H & E and then examined by light microscopy. The same paraffin slides were also processed for detection of TRAIL- and TUNEL-positive spots as described above. Tumour slides were also stained with a rabbit anti-E1A IgG Ab (Santa Cruz Biotechnology) and then treated with an ABC-peroxidase kit  to assess viral replication in tumour tissues.U87MG/Fluc orthotopic glioblastoma tumour model was prepared as described above using firefly luciferase-expressing U87MG cells for the evaluation of anti-tumour effect (n = 6). Seven days after the tumour cell injection, the mice were intracranially injected with PBS, H5CmTERT-Ad, or H5CmTERT-Ad/TRAIL . Statistical significance was determined by two-tailed Student’s T test . Data with Supplementary Figures


The tribological performances of nano-TiO2 lubricating fluid were measured by a four-ball tribotester. The hot rolling experiments under different lubrication conditions were carried out by a four-high rolling mill. The surface morphology, oxide scales and metallographic structure after hot rolling were observed using a confocal laser scanning microscope and scanning electron microscope (SEM), respectively. The composition of surface attachments was analyzed with X-ray photoelectron spectroscopy (XPS). The results indicate that the nano-TiO2 lubricating fluid has a better tribological performance. The surface defects on the hot rolled surface could be decreased. The phase composition of the surface still appears as a mixture of ferrite and pearlite. The surface of steel strips is not micro-alloyed with titanium as predicted. Additionally, the grain size of rolled steel strips which were lubricated with the nano-TiO2lubricating fluid decreased by nearly 50%, compared with traditional lubricating fluid. Furthermore, it was found that the thickness of the oxide layers on the surface reduced, whilst the Rockwell hardness of the oxide layers was enhanced as nano-TiO2 lubricating fluid was applied.In this paper, nano-TiO Recent 2) [2O3) ,11, mach2) [2O3) ,13,14, m2) [2O3) , etc. Pr process ,17.2 as an additive can improve the tribological properties of lubricants, few researchers have considered the impact of heating effect during the hot rolling process. Existing studies have principally focused on the effectiveness of nano-TiO2 on the rolling force, friction coefficient and other macro-performances. In this paper, the role of nano-TiO2 lubricating fluid on the microstructure of hot-rolled metal surface was investigated with interest. As the size decreases, anatase titanium dioxide can attain a more stable shape than rutile titanium dioxide at room temperature [2 nanoparticles used in nano-lubricating fluid are always in the anatase phase. The anatase phase is known to be metastable, while rutile is the stable phase. Therefore, the anatase phase will transform to the rutile phase under heat treatment at temperatures typically ranging between 600–900°C [2 are both octahedron structures consisting of titanium and oxygen elements. However, the fundamental structures of anatase nano-TiO2 are connected with the communal vertexes, while the fundamental structures of the rutile phase share the edges. Actually, there are some previous chemical bonds broken and some emerging chemical bonds formed during the heat treatment [2 nanoparticles used in nano-TiO2 lubricating fluid might have a chance to react with the surface steel during the hot rolling process and cause an inhomogeneity of the strips in structure and texture. The structural and textural inhomogeneity of steel strips would impact the processing properties and service performance of products [Although previous studies have verified that using nano-TiOperature  and henc00–900°C ,20. The reatment . Therefoproducts .2 lubricating fluid was prepared, and its lubrication performance was evaluated, using a four-ball tribotester. The surface morphology, oxidation, metallographic structures and elemental compositions on the surface of the SS41 steel strips, lubricated with or without nano-TiO2 lubricating fluid, were compared, to explore these problems. This exploration will play an important role in the application prospect of nano-TiO2 lubricating fluid used in hot rolling.In this paper, nano-TiO2  with a nominal diameter of 90 nm was used as the lubricant additive in this study. To prepare uniform and stable nano-fluid, the nano-TiO2 cannot be solely added into deionized water because of the polymerization of nanoparticles [2 lubricating fluid was prepared according to the formula shown in 2, was obtained as a control group. In this work, the traditional lubricating fluid was used to determine the interference of the other additives on the results.Anatase nano-TiOarticles . Both th3) used in the hot rolling test were provided by Jiuquan Iron & Steel (Group) Co., Ltd. The chemical compositions of the steel strips are listed in As the four-high rolling mill used in hot rolling test was an experimental mill with a working roller dimension of Ф170 × 350 mm, 70 mm was chosen as the breadth of the initial materials to reduce the rolling force. In addition, the steel strips , friction coefficient and wear scar diameter were measured on an MR-S10A four-ball tribometer, equipped with a rolling friction pair. The schematic of the four-ball rolling friction is shown in The tribological tests were taken to represent the tribological properties of nano-TiOB. While in the wear tests, the rotation velocity of the upper steel ball was 1200 r/min, the constant load was 392 N and the testing time was 30 min. The friction coefficients during the wear tests and the wear scar diameters after the wear test were measured to verify the friction reduction and antiwear behavior of nano-TiO2 lubricating fluid.A rotation velocity of 1760 r/min of the upper steel ball was applied in the representation of P2 lubricating fluid were used on the upper working roller in three-group comparative experiments, as shown in The austenite phase has lower strength and hardness than the ferrite phase —that is Four hot-rolled steel samples for each experimental group were prepared for analysis of surface morphology, oxidation, metallographic structure and surface chemical composition. The hot-rolled steel strips were cut into blocks of 10 × 10 mm from the center of the strips along the planes of the rolling direction (RD) and the normal direction (ND). The distance between each block was 20 mm.The rolled surface of the first samples can be directly used for surface morphology and roughness observations after rinsing with petroleum ether. To provide an intuitive description of the surface quality of the hot-rolled steel strips, the surface morphology after hot rolling was characterized by a confocal laser scanning microscope  for 2D and 3D topography observation. The linear roughness perpendicular to the rolling direction was measured. To ensure the accuracy of the data, each group of the samples was measured five times and the determined value was obtained by averaging five measurements. To distinguish the form of wear, SEM  was used for finer observation, and energy dispersive spectrometry  with a scanning electron microscope was applied to research the primary elementary composition of different forms of wear on the hot rolled surface.The second samples used for oxidation investigation had to be milled and polished to Ra < 0.1 μm. To explore thickness of the oxide layers, the cross-sectional blocks were ground with SiC paper of 240, 400, 800, 1500 and 2000 mesh, orderly, and then polished by nylon fabric with a 2.5 mm diamond polishing paste. The oxide layers were observed by SEM with backscattered electron microscopy (BSE) and the thickness of the oxide layers were measured by EDS with elemental linear scanning. In this work, the elemental linear scanning was taken on the cross-section, with the scanning direction perpendicular to the rolled surface. The higher oxygen content represented the behavior of the oxide layers, while the lower oxygen content represented the simple metal. The Rockwell hardness of the oxide scales was conducted on a hardness tester; the applied load and holding time were 100 kg and 60 s, respectively.The third samples were employed for the characterization of metallographic structure. The rolled surface of the samples had to be milled with SiC paper of 150 mesh to dislodge the oxide layers, and then milled and polished to Ra < 0.1 μm, using the same method mentioned in the last paragraph. After polishing, the surface was etched for 3–5 s with nitric acid alcohol, which was a mixture of 96% alcohol and 4% nitric acid, before microstructural analysis. The etched surface was surveyed by SEM, and the software of image analysis (Image Tool) was used to measure the grain size. To obtain a veracious datum, 12 adjoining grains in the image were chosen to be measured, and the average size was calculated, according to the 12 measurements. Then, the thicknesses of the samples were measured by micrometer and each final result was obtained by averaging six measurements.The last samples were cut into thin plates, each with a thickness of 1.5 mm, for the X-ray photoelectron spectroscopy , to determine the chemical states of typical elements on the strips’ surfaces.B) was signally enhanced by using TiO2 nanoparticles because of their remarkable film-forming property. This means the nano-TiO2 lubricating fluid can take effect under a greater positive pressure. The wear tests of each lubricant developed for 30 min, and the friction coefficients were measured and collected per second. The average friction coefficients of two lubricants were calculated to evaluate the friction reducing effect of using nano-TiO2. Wear scar diameters of the lower three steel balls were measured by optical microscope, and the mean values were calculated to evaluate the antiwear behavior of the nano-TiO2 lubricating fluid.The results of the tribology tests are shown in 2 fluid of the upper steel ball under 392 N at 1200 r/min are shown in 2 lubricating fluid, the friction coefficient was relatively consistent with the friction time. The results can exactly confirm the excellent film-forming property of nano-TiO2. Nano-TiO2 can easily adsorb to the steel surface to form a lubricating film. Thus, the friction coefficient-time curve of the nano-TiO2 lubricating fluid should be more stable.The friction coefficient-time curves of the traditional lubricating fluid and nano-TiO2 fluid was employed. It is known that optical microscopy has an inherently small depth field, whence the dark and bright regions can be associated with height difference. It indicates that the surface roughness of the hot-rolled strips would be decreased by using nano-TiO2lubricating fluid. These conclusions are confirmed by homologous 3D topographies in The optical micrograph and three-dimensional (3D) topography of hot-rolled strips under three different lubrication states were shown in 2 fluid, according to these results. Due to the adhesive wear, there must be some oxide scales and other hard materials adhered to the rolls. Therefore, the appearance of dense scratches parallel to the rolling direction is on account of scratching of the oxide scales and other hard materials adhered to the rolls. Comparing the size and depth of pits on three different surfaces, it is obvious that the pits on the non-lubricated surface are the largest, the pits in the traditional fluid lubricated surface take second place, and the pits in the nano-TiO2 fluid lubricated surface are smallest and shallowest. As a consequence, the scratches in The SEM was used to distinguish and analyze these surface defects. The SEM images are shown in As shown in 2 fluid was used, the oxide scales become compact and fewer voids can be seen in the SEM image. The Rockwell hardness’ of the oxide scales listed in the top right corner of the SEM images are consistent with the visual results—the more compact the oxide scales are, higher the hardness is. The EDS with elemental linear scanning was taken to measure the thickness of the oxide scales, and the scanning direction is annotated on the SEM images. The vertical distributions of element Fe and element O in the transverse section were quantitated, and the spots whose oxygen content was above zero were considered to be the oxide scales. The thickness of the oxide layers without any lubrication was about 40 μm ; the results are shown in 2 nanoparticles are added into the lubricant, and the overall reduction in the same rolling period can be enhanced visibly. On account of the rise of rolling deformation, more and more dislocations and lattice defects appeared. As a result, the nucleation rate increased due to the enhancement of lattice distortion energy, and the grains became refined [The main reason for this phenomenon is the increase in rolling deformation. The finishing thicknesses and spring-back values of hot rolled steel strips were shown in  refined .2 was added, the crystal structure of nano-TiO2 and oxide scales were analyzed. The element Fe on the surfaces exists in the forms, Fe3+ and Fe2+, and the titanium in nano-TiO2 is Ti4+. According to the crystal structures of TiO2, Fe2O3 and FeO  is close to that of Fe3+ (0.68 Å) [2+ (0.78 Å) and Ti4+ (0.64 Å) have great differentials, so it is not easy for Ti4+ to pass into the crystal lattice of Fe3O4.To explore the reason why no new phase appeared as the nano-TiO and FeO , the lig(0.68 Å) . On the 2O3 which has a loose structure; the innermost layer is FeO which is denser, and the interlayer is Fe3O4 which is a mixture of Fe2O3 and FeO. Though there might be some dissociative Ti4+ which can pass into the surface, this would exist in Fe2O3, which is a porous oxide scale covered on the steel surface. The TiO2 nanoparticles would adsorb on the hole of the oxide scale; as a consequence, there is only a chemical adhesion film created on the oxide scales, but no new phase appeared in the metal base.There are three kinds of iron oxides covered on the surface of steel strips. The outermost layer is Fe2 lubricating fluid. The following conclusions were drawn from this study:(1)2 in lubricating fluid was associated with a 62.5% increase in the maximum non-seizure load, a 33.8% reduction in the friction coefficient and a 47.4% reduction in the wear extent, compared with traditional lubricating fluid. Moreover, the friction coefficient-time curve of nano-TiO2 lubricating fluid became smooth in only 50 s, while that of traditional lubricating fluid presented a remarkably increasing trend till 900 s.Adding nano-TiO(2)2 lubricating fluid decreased from 18.51 μm to 7.46 μm. Additionally, the phase composition of the surface still appeared as a mixture of ferrite and pearlite. There was no iron micro-alloyed phenomenon with titanium. The homogeneity of the metal base was not destroyed.The grain size of rolled steel strips lubricated with nano-TiO(3)2 nanoparticles could deposit on worn surfaces in the hot rolling process. With the generation of iron oxides, nano-TiO2 was distributed in the oxide scales diffusely. The compactness of the oxide scales could be drastically enhanced, which caused a slightly increase in Rockwell hardness and a decrease in the thickness of the oxide scales, from 60 μm to 20 μm. There were fewer surface defects on the hot rolled surface.TiOThe present work analyzed the surface morphology and metallographic structure of hot rolled SS41 steel, lubricated with nano-TiO


Many efforts have been made to predict, before giving fluid, whether it will increase cardiac output. Nevertheless, after fluid administration, it is also essential to assess the therapeutic efficacy and to look for possible adverse effects. Like for any drug, this step should not be missed. Basically, volume expansion is aimed at improving tissue oxygenation and organ function. To assess this final result, clinical signs are often unhelpful. The increase in urine output in case of acute kidney injury is a poor marker of the kidney perfusion improvement. Even if oxygen delivery has increased with fluid, the increase in oxygen consumption is not constant. Assessing this response needs to measure markers such as lactate, central/mixed venous oxygen saturation, or carbon dioxide-derived indices. If tissue oxygenation did not improve, one should check that cardiac output has actually increased with fluid administration. To assess this response, changes in arterial pressure are not reliable enough, and direct measurements of cardiac output are required. In cases where cardiac output did not increase with fluid, one should check that it was not due to an insufficient volume of fluid administered. For this purpose, volume markers of cardiac preload sometimes lack precision. The central venous pressure, in theory at least, should not augment to a large extent in fluid responders. The worst adverse effect of fluids is the increase in the cumulative fluid balance. In patients with acute respiratory distress syndrome (ARDS), the risk of aggravating pulmonary oedema should be systematically assessed by looking for increases in extravascular lung water, or, more indirectly, increases in central venous or pulmonary artery occlusion pressure. In ARDS patients receiving fluid, one should always keep in mind the risk of inducing/aggravating right ventricular dilation, which should be confirmed through echocardiography. The risk of increasing the intra-abdominal pressure should be carefully sought in patients at risk. Finally, fluid-induced haemodilution should not be neglected. Like for any drug which has inconsistent effectiveness and may exert significant harm, the correct fluid management should include a cautious and comprehensive assessment of fluid-induced benefits and side effects. Despite its trivial aspect, administration of fluid in critically ill patients poses several complex problems. Many efforts have been made to determine, before giving fluid, whether it will increase cardiac output. Nevertheless, after fluid administration, like for any treatment which has inconsistent effectiveness and which might induce significant side effects, it is essential to ask: was volume expansion effective? Was it harmful? Fluids are drugs. Assessing their efficiency and their adverse side effects should be part of good clinical practice. It seems to us that this step is often missed. In this review, based on the published data, we will attempt to show how essential it is to carefully evaluate the positive and negative effects of volume expansion once it has been administered and which evaluation criteria might be useful for this purpose.When fluid is administered in case of acute circulatory failure, it is with the final goal of increasing tissue oxygenation and, if it was previously impaired, of improving organ function. Nevertheless, between volume expansion and the resolution of organ failure, multiple steps are to be crossed Fig. . At eachBasically, fluid is administered in order to increase the mean systemic pressure, which is the forward pressure of venous return. Nevertheless, capillary leak or vasodilation may impede that fluid actually increases the stressed blood volume . If it oThe increase in arterial flow and/or pressure increases the blood flow through the microcirculation and, sometimes, this is accompanied by a significant increase in the proportion of perfused vessels . This stFinally, the reduction in tissue hypoxia should improve organ function. Nevertheless, this becomes impossible if organ function has been structurally injured by prolonged hypoperfusion. For instance, tubular necrosis explains that acute kidney injury persists in spite of resolution of the acute phase of shock Fig. .Basically, the decision to infuse fluids should be triggered by obvious signs of tissue hypoperfusion. In particular, it should never be taken only for increasing cardiac output, for instance, on the basis of positive tests of fluid responsiveness. The harmful effects of fluid infusion are today clearly demonstrated, and strategies aiming at systematically maximising oxygen delivery and cardiac output have been shown to be useless or even deleterious .The resolution of organ dysfunction that has been induced by shock, like acute kidney injury, obviously indicates that tissue perfusion occurred. Nevertheless, as stated above, the increase in urine output is unhelpful in the numerous cases in which tubular necrosis has already occurred. Even if this is not the case, the increase in diuresis is very poorly correlated with the simultaneous changes in cardiac output . MoreoveIn general, looking for the improvement in tissue oxygenation likely requires more sophisticated indices Fig. . In a stThese results do not mean that volume expansion was unnecessary in cases where oxygen consumption did not increase. The increase in oxygen delivery profitably moves the patient away from the dangerous zone where oxygen consumption becomes dependent on delivery. Nevertheless, it must be kept in mind that aiming at achieving supranormal values for cardiac output or normal values for mixed venous oxygen saturation does not reduce morbidity or mortality among critically ill patients .2 and ScvO2, respectively) might be useful. When it is low and when it increases with volume expansion, it means that tissue oxygenation improved . Thi. Thi1]. In order to rule out the latter hypothesis, one may look at the fluid-induced changes in the markers of cardiac preload. Among them, the cardiac end-diastolic volume might be estimated through echocardiography or transpulmonary thermodilution . In theoThe measurement of central venous pressure is more precise. Nevertheless, central venous pressure does not necessarily increase significantly, even in cases where volume expansion has increased cardiac preload. Indeed, in patients who respond to volume expansion by an increase in cardiac output—and thus in venous return, which equals cardiac output at steady state—the increase in venous return is provoked by an increase in its pressure gradient . For this to happen, right atrial pressure—and thus central venous pressure—must increase to a lesser extent than the mean systemic pressure. Thus, in theory, fluid administration might be effective and cardiac output might increase although the central venous pressure did not change to a large extent. This limitation might be overcome by measuring the mean systemic pressure, but this is not possible in routine practice. Nevertheless, it remains that in fluid non-responders, central venous pressure increases as much as the mean systemic pressure. Then, in theory, if cardiac output does not increase with volume expansion, a reliable way to ascertain that this was due to fluid unresponsiveness, and not to an insufficient fluid volume, is to look for an increase in the central venous pressure. Nevertheless, increasing the central venous pressure is not per se a goal of fluid administration.Like any other drug, whatever its efficacy, the adverse side effects that may result from fluid administration must be carefully searched. Continuously balancing their weight against haemodynamic benefits helps decide to go on with fluid administration or to choose another therapeutic strategy.Volume expansion might increase the total cumulative fluid balance in case of renal failure. As it has been now very clearly established, the higher the cumulative fluid balance, the higher the mortality of critically ill patients, especially in case of acute respiratory distress syndrome (ARDS)  and/or sBesides the cumulative fluid balance, more specific signs of fluid overload must be sought. An increase in the peripheral subcutaneous oedema clearly indicates that fluid has leaked out of the vessels. Obviously, the highest risk is to aggravate lung oedema. Chest X-ray is not sensitive enough since it can detect only large increases in lung oedema. High values of central venous pressure or of pulmonary artery occlusion pressure could be more specific. Nevertheless, they do not take into account a key parameter, which is pulmonary capillary permeability. If pulmonary capillary permeability is much increased, lung oedema might have appeared even though the values of pulmonary artery occlusion pressure and central venous pressure are not much increased.Extravascular lung water, which is the volume of fluid accumulated in the interstitium and alveoli, is a much more direct reflection of lung oedema created by fluid accumulation Fig. . Then, iIn case of right ventricular failure, as during ARDS, a specific risk of fluid infusion is to aggravate the right ventricular dilation. The right ventricle is very sensitive to changes in its afterload, which explains the risk that it dilates in case of ARDS. In addition, in case of right ventricular preload unresponsiveness, any fluid administration might further increase the right ventricular overload, and finally the right ventricular dilation. The septal shift it induces should reduce the left ventricular filling and contribute to the decrease in cardiac output.The right ventricular impairment should be assessed if several fluid infusions have been repeated in a few hours. There is no doubt that echocardiography is the gold standard to assess the right ventricular failure. Nevertheless, it cannot be easily repeated over the day. A faster and easier means is to look for increases in the central venous pressure . This emWorsening of prior intra-abdominal hypertension (IAH) is likely one of the adverse effects of fluid resuscitation that is often neglected in practice. One should always keep in mind that the incidence of IAH in critically ill patients is around 20–30% on admission, while as many as 50–70% of patients (depending on the condition) may develop IAH during the first week of stay in the intensive care unit .One of the most important risk factors for IAH is fluid resuscitation . There iAdministration of crystalloids or colloids unavoidably results in haemodilution, and the degree of this haemodilution is far from negligible. One must keep in mind that the normal total blood volume is 65–70 mL/kg and that it is even reduced in many conditions associated with circulatory failure. A volume expansion of even only 500 mL is significant.In a study in which critically ill patients received a 500-mL volume expansion, our group showed that this resulted in a decrease in haemoglobin concentration by 8%. More importantly, in patients who did not respond to fluid by an increase in cardiac output (non-responders), this led to a significant decrease in oxygen delivery . ClearlyFluids should be considered as a drug, which positive effects are inconstant and which carries a significant risk of adverse effects. Like for any drug, one should not miss after having administered fluids to ask two questions: has it been effective and has it been harmful? Who would administer antibiotics without assessing the body temperature or the biological markers of inflammation? Who would administer aminosides without checking the renal function? The correct fluid management should not be limited to the prediction of fluid responsiveness, but should include a cautious assessment of fluids benefits and side effects.


Recently published study of Ma et al. evaluates two relatively novel measures of fluid responsiveness, carotid blood flow and corrected carotid flow time (ccFT). Both measures have been recently quoted as possibly useful, technically simple, and noninvasive dynamic tools in predicting fluid responsiveness. Recently, more research interest has been focused on ccFT and, intrigued by the data presented in this study, we discuss here the impact of the data presented in the paper of Ma et al. to the significance of this metric as a potential tool in the assessment of fluid responsiveness. Dear Editor,It was a great pleasure to read about the study of Ma et al.  which evThe study of Ma et al. sheds a new light on the role of ccFT and is the first prospective clinical study to compare ccFT with the gold standard of cardiac output measurement, right heart catheterization (RHC). This study evaluated patients undergoing diagnostic RHC who were subjected to a passive leg raise maneuver (PLR) which mimics a fluid bolus and has been used to assess fluid responsiveness in shock . Unfortuchanges in this measures that may correlate better. Several studies have looked at the change of ccFT (∆ccFT) as a potential tool in the process of evaluation of fluid responsiveness and found that change in duration of ccFT in dehydrated patients receiving intravenous fluid resuscitation [In this study, ccFT did not correlate well with cardiac output which is not surprising. Flow time is a proxy for left ventricular ejection time, which has classically been considered to represent a static hemodynamic measure, along with other “static” preload indices , 7, and citation , 8, 9 orcitation  may be hcitation . Practiccitation .There are several methodological concerns worth bringing up in order to separate a moderate correlation of average ccFT and cardiac output presented in the study of Ma et al. and possible usefulness of the change in ccFT in fluid resuscitation of patients in shock. Patients undergoing RHC who were evaluated in the study of Ma et al. may have very little in common with the population of fluid under-resuscitated patients in shock who are usually evaluated with PLR and where the role of ccFT has been recently discussed as possibly relevant . The cohFrom the technical perspective, an interesting idea presented in the study of Ma et al. is an attempt to avoid the selection bias by averaging ccFT values of multiple (three) heart beats instead of taking a single value. Understanding the impact of breathing on cardiac rhythm, this concept is valid and important but the method may require further optimization as averaging should probably be taken in the context of respiratory cycle rather than randomly selecting three beats.There is a common agreement that the dynamic parameters have become a gold standard in fluid responsiveness assessment , but the


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Genetic variations have been identified in the genome of varicella-zoster virus (VZV) strains using vesicle fluid, varicella scabs and throat swab samples. We report a rare case of VZV-associated uveitis with severe hyphema, which was immediately diagnosed by polymerase chain reaction (PCR) using the aqueous humor, in which we were able to analyze the VZV genotype for the first time.9 copies/mL of VZV-DNA were detected by real-time PCR using the aqueous humor. As there were no eruptions on his face or body, we diagnosed zoster sine herpete and started intravenous administration of prednisolone and acyclovir. The hyphema completely disappeared 2 weeks after presentation, while sectorial iris atrophy and mild periphlebitis of the fundus became gradually apparent. Anterior inflammation and periphlebitis gradually improved and VZV-DNA in the aqueous humor was reduced to 1.02 × 106 copies/mL at 4 weeks after presentation. Examination by slit lamp microscope revealed no inflammation after 5 months, and VZV-DNA could no longer be detected in the aqueous humor. Serum anti-VZV IgG and anti-VZV IgM also showed a gradual decrease along with improvement in ocular inflammation. The genetic analysis of multiple open reading frames and the R5 variable repeat region in the VZV genes, using DNA extracted from the aqueous humor at presentation, showed that the isolate was a wild-type clade 2 VZV strain  with R5A allele and one SNP unique to clade 1 (both are major types in Europe and North America).A 16-year-old Japanese boy was referred to our hospital with a 20-day history of unilateral anterior uveitis and 11-day history of hyphema. At presentation, details of the iris, the iridocorneal angle, and the fundus were not visible due to the severe hyphema. Serum anti-VZV IgG and anti-VZV IgM were elevated, and 1.61 × 109 copies/mL).VZV-associated uveitis may develop hyphema that obscures ocular inflammation, thus PCR analysis using the aqueous humor is the key investigation necessary for the diagnosis. The measurement of VZV-DNA copies by real-time PCR would be useful for evaluation of therapeutic effects. We could amplify and analyze VZV genotype using the aqueous humor including a very large number of VZV-DNA copies (1.61 × 10 Varicella-zoster virus (VZV) is highly contagious and globally ubiquitous. The primary infection results in varicella (chickenpox), which usually occurs early in life. Subsequently, the virus establishes a lifelong latent infection in the sensory nerve ganglia, which reactivates to cause herpes zoster .The VZV genome consists of about 125,000 bases of linear, double-stranded DNA with ≧ 70 open reading frames (ORFs). Since the 1990s, a number of genetic variations have been identified in the genome of VZV strains by the use of molecular techniques such as sequencing, restriction fragment length polymorphism (RFLP) and single nucleotide polymorphism (SNP) . In thesHerpes zoster involvement in the ophthalmic division of the first branch of the trigeminal nerve is called herpes zoster ophthalmicus (HZO). HZO without skin lesions is known as zoster sine herpete (ZSH). VZV-associated uveitis may develop in both HZO and ZSH . VZV antHere, we report a rare case of VZV-associated uveitis with severe hyphema, early diagnosed by multiplex polymerase chain reaction (PCR) using the aqueous humor, in which we were able to amplify and analyze the VZV genotype for the first time.A 16-year-old Japanese boy was referred to our hospital from an ophthalmology clinic because of severe hyphema in the left eye and anterior uveitis that had persisted for 11 and 20 days, respectively. He was receiving topical treatment with 0.1% betamethasone, 1% atropine, 1.5% levofloxacin, and 0.5% timolol maleate in the left eye. There was no ocular history of trauma, inflammation, or medication. His family history and medical history for systemic diseases were unremarkable. At presentation, the best corrected visual acuity was 20/15 in the right eye and 20/2000 in the left eye. Intraocular pressure (IOP) was 12 mmHg in the right eye and 29 mmHg in the left eye. Slit lamp biomicroscopy of the left eye revealed ciliary injection, corneal edema, and severe hyphema filling the anterior chamber , anti-nuclear antibody, or rheumatoid factor. However, anti-VZV IgG and anti-VZV IgM measured by enzyme-linked immunosorbent assays were elevated to >2600 mIU/mL (negative: < 50 mIU/mL) and 1: 672 (negative: < 1: 42), respectively. VZV-DNA was not detected in the plasma.9 copies/mL of VZV-DNA. He had not been vaccinated for VZV and there was history of chickenpox in early childhood. As there were no skin eruptions on his face or body, we diagnosed ZSH and excluded HZO. A detailed examination of the fundus was impossible because of hyphema, so we initiated intravenous administration of acyclovir (500 mg/day) and dexamethasone (6.6 mg/day) based on the possibility of acute retinal necrosis.In addition to the laboratory blood tests, we performed an anterior chamber paracentesis of the left eye and extracted DNA from the aqueous humor sample using QIAamp DNA Mini Kit . To detect human herpes viruses 1 through 6, multiplex PCR was also performed. VZV-DNA was then detected by multiplex PCR and quantitative real-time PCR revealed 1.61 × 10The hyphema was gradually absorbed and visibility of the anterior chamber improved, revealing inflammation of the anterior chamber and distortion of the pupil Fig. . A subcoAt two weeks after the initial visit, the hyphema had completely resolved and segmental iris atrophy, which is one of the characteristic ocular manifestations of VZV-associated uveitis, was visible Fig. . Visual 6 copies/mL. At 2 months after presentation, there was no deterioration in ocular inflammation, so oral famciclovir was reduced to 750 mg/day. At 3 months after presentation, the ciliary injection, anterior inflammation, and periphlebitis had become very mild, and the serum anti-VZV IgG and anti-VZV IgM were further decreased to 1300 mIU/mL and 1: 42, respectively. Treatment was continued with oral famciclovir (750 mg/day) and prednisolone (5 mg/day).At 4 weeks after the initial visit, anti-VZV IgG and anti-VZV IgM had decreased to 2500 mIU/mL and 1: 168, respectively. VZV-DNA in the aqueous humor was reduced to 1.02 × 10At 4 months after presentation, as the ciliary injection and inflammation of the anterior chamber had worsened and rubeosis iridis emerged Fig. , a subteAt 5 months after presentation, visual acuity was 20/25 and the IOP was 10 mmHg in the left eye. The anterior inflammation and periphlebitis had almost completely resolved and the rubeosis iridis had disappeared Fig. . Slit la14 months after the initial visit, visual acuity was 20/40 and the IOP was 12 mmHg in the left eye. There was no recurrence of severe inflammation, but a cataract had gradually developed. Anterior segment optical coherence tomography (OCT) images obtained from the CASIA  revealed thinning of the iris in the left eye Fig. . Serum a9 copies/mL of VZV-DNA). Attempts to sequence for VZV genotyping using DNA obtained at 4 weeks after presentation (1.02 × 106 copies/mL) were unsuccessful due to the low viral load. First, we analyzed the RFLPs of ORF38 (PstI), ORF54 (BglI), and ORF62 (SmaI) as previously described [PstI restriction enzyme site in ORF38 (positive for the PstI site: PstI+). In this case, the PCR products of ORF38 (647 bp) lacked this restriction site (negative for the PstI site: PstI−)  and yielded 2 fragments of 256 and 241 bp  [SmaI site: SmaI−) like most wild-type VZV strains , positive for the BglI site (BglI+), and negative for the SmaI site (SmaI−), which are typical of Japanese wild-type strains such as the parental Oka (p-Oka) strain and strain Kawaguchi  Fig. . The PCR bp Fig. . The PCRins Fig. . In summThe R5 variable region between ORF60 and ORF61 has also been shown to vary among different VZV strains. The R5 variable region was amplified by PCR and analyzed by electrophoresis as previously described . The sizn = 1), those of the R5B type are 471 bp (n = 2), and those of the R5C type are 583 bp (n = 3). In this patient, R5A (mainly found in Europe and North America) was observed unlike Japanese strains   in the aqueous humor of eight patients with VZV-associated uveitis [9 copies/mL) in the aqueous humor at presentation, which subsequently fell to 1.02 × 106 copies/mL at 4 weeks after presentation. We could analyze the VZV genotype using DNA obtained at presentation, but not with DNA obtained at 4 weeks after presentation. This is a valuable report in terms of our ability to obtain a very large number of VZV-DNA copies and analyze VZV genotype using the aqueous humor. To the best of our knowledge, this is the first investigation of VZV genotype to use aqueous humor.Genetic variations have been identified in the genome of VZV strains from vesicle fluid, varicella scabs or throat swab samples by molecular techniques –9. Howev uveitis . In thisPstI), ORF54 (BglI) and ORF62 (SmaI) in the VZV genes are molecular genetic markers for the genotyping of VZV strains [PstI restriction site (PstI−) in ORF 38 and the presence of a BglI restriction site (BglI+) in ORF 54 differentiate v-Oka from wild-type American strains [SmaI restriction enzyme cleavage sites in ORF 62, which only v-Oka creates, v-Oka can be differentiated from p-Oka and other wild-type Japanese strains [PstI− in ORF38, BglI+ in ORF54, and SmaI− in ORF 62  and two provisional clades (VI and VII) were proposed . Multipl and two As there have been no reports on the genotyping of VZV in uveitis using aqueous humor, it is unknown whether this genotype is specific to uveitis or whether it differs from varicella or herpes zoster. Despite the thorough investigation of genetic variability among the circulating strains of VZV in recent decades, it is not yet known whether VZV genotype is linked to differences in the pathogenesis and virulence of VZV strains. One of the important limitations to this report is that we were not able to analyze any other VZV-DNA sequences from this patient to which the aqueous humor genomes could be compared as VZV-DNA was not detected in the plasma and no other specimens, such as throat swabs, could be collected at presentation. Therefore, future studies are warranted to analyze VZV genotypes using the aqueous humor, as performed in this study, as well as other specimens where possible in a large number of uveitis cases.9 copies/mL). Genetic analysis showed that the isolate from the aqueous humor of this patient was a wild-type clade 2 VZV strain  with R5A allele and one SNP unique to clade 1 (both are mainly found in Europe and North America).In conclusion, we report a rare case of ZSH with severe hyphema, which was diagnosed by multiplex PCR using the aqueous humor at an early stage. The measurement of VZV-DNA copy numbers by quantitative real-time PCR, as well as serum anti-VZV IgG and serum anti-VZV IgM was useful for the evaluation of improvement in ocular inflammation and therapeutic effects. As far as we know, this is the first report that we could amplify and analyze VZV genotype using the aqueous humor including a very large number of VZV-DNA copies (1.61 × 10


Primary varicella zoster virus (VZV) infection, predominantly in the pediatric population, presents with pyrexia and a classic pruritic vesicular rash. In adults, although less common, it is more severe and linked to more complications. Neurological complications, which account for less than 1% of all VZV complications, include meningitis, encephalitis, arterial vasculopathy, and venous thrombosis. We present a case of a 39-year-old male who developed extensive cerebral venous sinus thrombosis following primary VZV infection. Venous thrombosis in VZV has been suggested to be caused by autoantibodies against protein S, pre-existing hypercoagulability, or endothelial damage. The patient was acutely managed using intravenous acyclovir and heparin. Long-term anticoagulation therapy with warfarin was continued after discharge. We concluded that clinicians should be aware of the rare complications of this common pathology so that a timely diagnosis can be made, followed by prompt management. Further studies need to be done to better understand acute cerebral venous sinus thrombosis secondary to VZV. Primary varicella zoster virus (VZV) classically presents as a febrile illness in children with an exanthematous vesicular rash. The epidemiology of VZV is influenced by climate. In temperate climates, the incidence of VZV is 13-16 cases per 1,000 people per year. Greater than 90% of those are infected before adolescence. In tropical climates, VZV tends to occur later in life and adults are more prone to be affected than children -3.Very rarely, cerebral venous thrombosis secondary to primary VZV infection can occur due to a prothrombotic state . We repoA 39-year-old male with a known case of chickenpox (primary VZV infection) of seven days duration presented at the emergency department (ED) with complaints of a headache and vomiting. The dull aching headache started five days prior in the occipital region, radiating to the neck, and was moderate to severe in intensity with no aggravating or relieving factors. Vomiting began two days prior, with six episodes per day, and no relief with medicines. The patient also had associated anorexia, lethargy, dizziness, and intermittent fever, varying between 99° F - 102° F for five days. No photophobia, shortness of breath, palpitations, or visual disturbances were noted. Prior medical, surgical, and family history were insignificant. Chickenpox, which started a week prior, was diagnosed on the basis of history and the clinical exam.On initial examination, the patient was dehydrated and in discomfort. Scattered exanthematous vesicular lesions were observed all over his body, particularly on the face and trunk. His temperature was 100° F with a pulse of 70 beats per minute, blood pressure of 140/90 mmHg, respiratory rate of 18 breaths per minute, and a SpO2 of 97%. The patient's Glasgow Coma Score (GCS) was 15/15 and positive for slight neck stiffness; however, no weakness or neurological deficits were observed. The patient's strength was 5/5 in all four limbs with a complete range of motion, normal deep tendon reflexes, and a negative Babinski sign. In the ED, further workup was done, which included serologic and radiologic investigations. A computed tomography (CT) scan revealed extensive cerebral venous sinus thrombosis, as shown in Figures By day three, the patient started improving and there were no more active complaints of a headache, vomiting, or fever. The patient was also started on an adjusted dose of warfarin for long-term anticoagulation until a therapeutic international normalized ratio (INR) was attained. Intravenous acyclovir was discontinued after the course was completed, and heparin was bridged to warfarin for long-term anticoagulation. After a week of no active complaints, the patient was discharged in a stable condition with long-term anticoagulation therapy and a follow-up plan of care. The patient was also advised to return to the ED in case of any alarming symptoms in the future.Primary VZV infection, also known as chickenpox, is a self-limiting febrile illness causing an exanthematous vesicular rash most commonly seen in children. Adults usually have reactivation of VZV as compared to primary varicella infection. Complications associated with VZV include encephalitis, cerebellar ataxia, transverse myelitis, ventriculitis, meningoencephalitis, and aseptic meningitis . It can The basis of venous thrombosis can be linked to Virchow's triad, which includes injury to the vessel walls, a hypercoagulable state, and stasis . The mecCVST can present with isolated intracranial hypertension causing headache, vomiting, and papilledema. It may also present with convulsions, altered mental status, cranial nerve palsies, or focal deficits . Our patVasculopathies proceeding after VZV infection are often diagnosed using a cerebrospinal fluid polymerase chain reaction for anti-VZV immunoglobulin G antibodies and VZV deoxyribonucleic acid (DNA). Unless both are negative, vasculopathy cannot be ruled out . If CVSTVirus-associated vascular complications are frequently treated using intravenous acyclovir . FurtherWe conclude that the clinician should be aware of rare complications and their mechanisms so that a timely diagnosis can be made and prompt treatment can be initiated. Patients with primary VZV infection are prone to hypercoagulable states, owing to a decrease in natural anticoagulant levels. As a consequence, a fatal widespread thromboembolic phenomenon can occur. Prompt treatment with adequate hydration and anticoagulation can reduce mortality and improve the prognosis. Further studies need to be done to improve the understanding of CVST as a complication of primary VZV infection.


Metaplastic breast carcinoma (MBC) is a rare subtype of invasive breast cancer and has poor prognosis. In general, cancers are heterogeneous cellular masses comprised of different cell types and their extracellular matrix (ECM). However, little is known about the composition of the ECM and its constituents in MBC. Decorin is a ubiquitous ECM macromolecule known of its oncosuppressive activity. As such, it provides an intriguing molecule in the development of novel therapeutics for different malignancies such as MBC. In this study, decorin immunoreactivity and the effect of adenoviral decorin cDNA (Ad-DCN) transduction were examined in MBC. Multiple immunohistochemical stainings were used to characterize a massive breast tumour derived from an old woman. Furthermore, three-dimensional (3D) explant cultures derived from the tumour were transduced with Ad-DCN to study the effect of the transduction on the explants. The MBC tumour was shown to be completely negative for decorin immunoreactivity demonstrating that the malignant cells were not able to synthesize decorin. Ad-DCN transduction resulted in a markedly altered cytological phenotype of MBC explants by decreasing the amount of atypical cells and by inhibiting cell proliferation. The results of this study support approaches to develop new, decorin-based adjuvant therapies for MBC.The online version of this article (doi:10.1007/s12307-017-0195-8) contains supplementary material, which is available to authorized users. Metaplastic breast carcinoma (MBC) is a rare subtype of invasive breast cancer accounting for 0.2–5% of all breast malignancies . MBC is MBC is usually associated with a lower incidence of axillary nodal involvement compared to typical breast carcinomas of similar size . NeverthIn general, cancers are heterogeneous cellular masses comprised of various cell types including malignant cells, immune cells, epithelial cells, stromal cells and their extracellular matrix (ECM) , 15. CelCertain ECM macromolecules, particularly proteoglycans (PGs) such as decorin, have variously been identified as one of the key players capable of modulating cell signaling, adhesion, migration, proliferation, and apoptosis , 28. DecIn this case report, a massive breast tumour was first characterized as MBC. Next, the MBC tumour was examined for immunoreactivity for the above two stromal PGs, decorin and biglycan. Thereafter, 3D MBC explant cultures were established and the effect of Ad-DCN transduction on the cytology and proliferation index of the explants was examined. Finally, the effect of Ad-DCN transduction on the expression of the ErbB family members, EGFR (ErbB1), ErbB2 , ErbB3 and ErbB4 was also analyzed.We present a case of an 87 years old Finnish woman, who was admitted to Turku University Hospital, Turku, Finland, with a locally advanced breast cancer with ulceration. She was in moderate condition for her years. Her breast examination revealed breast tissue that was red, swollen and had a 3–4 cm ulceration with cystic cavity. The palpable tumour was over 10 cm in diameter. The study protocol was approved by the Joint Ethics Committee of the University of Turku and Turku University Hospital, Turku, Finland (number TO6/029/15). Informed consent was obtained from the patient.After radical mastectomy, a representative sample from the fresh breast tumour tissue (BTT) was obtained within half an hour of surgery. The tissue sample was taken from the invasive border of the tumour by a pathologist. Thereafter, the tumour tissue was divided into samples for the immunohistochemistry (IHC) analyses, and samples for the 3D transduction experiments.ultraVIEW Universal DAB Detection Kit . All the above listed antibodies were ready-to-use dilutions . Antibodies against androgen receptor  and gross cystic disease fluid protein-15  from Novocastra  were also used. The antibodies for AR and GCDFP-15 were diluted in Antibody Diluent from Ventana .The BTT samples for hematoxylin and eosin (HE) staining and IHC were fixed in 10% neutral-buffered formalin, embedded in paraffin and cut into 4 μm consecutive sections. The sections were stained with antibodies against ER , PR , HER2 , EGFR , cytokeratin Pan , cytokeratin 7 , p63 , CD10 , vimentin , epithelial membrane antigen , CK5/6  and Ki-67  using Ventana Medical Systems/Roche Diagnostics with BenchMark XT immunostainer and Additionally, tumour sections were examined for immunoreactivity for decorin (DCN) and biglycan (BGN) . Briefly2 and 95% air at +37 °C. After 24 h, the tumour explants were transduced as described below.The BTT samples were transported to the cell culture laboratory in cold (+4 °C), sterile 0.9% sodium chloride (NaCl) supplemented with penicillin 100 IU/mL), streptomycin (100 μg/mL) and Fungizone . The 3D explant cultures were established as previously described with some minor modifications –55. Brie00 IU/mL,4 cells in horizontal surface of the tissue section. In negative control cultures the medium was substituted with fresh one. After 24 h, the medium in the wells was replaced with fresh one. Next day, tissue explants and dental sponges underneath them were fixed in 4% paraformaldehyde for 48 h at +4 °C and embedded in paraffin. The paraffin blocks were cut into 4 μm consecutive sections and stained for HE and Ki-67 to evaluate the effect of Ad-DCN transduction on the cytology and the proliferation index of the explants.The 3D MBC explant cultures were transduced with a recombinant replication-deficient adenoviral vector carrying human decorin cDNA (Ad-DCN) as previously described  with minhttp://153.1.200.58:8080/immunoratio/). The program automatically calculates the ratio of Ki-67 positive cells to total cell count.All tissue sections were scanned with Pannoramic Digital Slide scanner . Digital images were viewed with Pannoramic Viewer (3DHISTECH). Nuclear staining <1% of the tumour cells was considered negative for ER and PR. HER2 expression was evaluated as membrane staining of invasive tumour cells according to American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines . CK5/6 aTotal RNA was extracted from the 3D MBC explant cultures using TRIsure RNA isolation reagent . To eliminate possible contaminating DNA, RNA samples were treated with 10 units of DNase I (Roche). cDNA was synthesized in a reaction using 1 μg of total RNA and Sensifast cDNA Synthesis Kit (Bioline) according to the manufacturer’s protocol.Real-Time PCR (RT-qPCR) analyses of EGFR, ErbB2, ErbB3 and ErbB4 (JM-a) isoform expression was performed as previously described . Expressp values <0.05 were considered statistically significant.The effect of Ad-DCN transduction on the proliferation index of the tissue explants was evaluated using Tukey’s Multiple Comparison Test. The Gross examination of the mastectomy specimen revealed a necrotic, cystic and haemorrhagic tumour mass measuring over 10 cm in diameter with poorly circumscribed infiltrative margins Fig. . There wThe histopathology showed that the tumour was composed of intersection bundles of pleomorphic spindle shaped cells with large pleomorphic hyperchromatic nucleus and moderate amount of cytoplasm. There were also round to polygonal cells arranged in irregular sheets and occasional ductal structures. Apocrine differentiation was found focally with various clusters of papillary projections and sheets. In addition, a small, irregular lace-like osteoid was seen. Mitotic figures were numerous (data not shown).The immunohistological profile of the tumour revealed a neoplasm with a highly heterogeneous histology. Selected IHC images are shown in Fig. Next we examined the immunoreactivity for two stromal PGs, namely DCN and BGN, in this MBC tumour. The results demonstrated that the tumour tissue was completely negative for DCN immunoreactivity, while BGN immunoreactivity was locally clearly detected Fig. .Fig. 4ImLight microscopic observation of 3D MBC explant cultures revealed that Ad-DCN transduction resulted in a marked decrease in the cellularity and in the amount of atypical stromal cells compared to control vector (Ad-LacZ) transduced explants and to negative control cultures Fig. . ExplantThe expression of the ErbB family members in the transduced BTT samples was studied with RT-qPCR. The results indicated that the MBC tissue expressed roughly similar levels of both EGFR and ErbB2 in all the samples; decorin cDNA and LacZ transduced, and negative controls , this increase in fibroblast-like cells is most likely the result of decorin’s capability to normalize the tumour tissue histology rather than to promote more aggressive mesenchymal phenotype typical for MBC –70. NeveAlso the proliferation index of cancer tissue was statistically significantly decreased in response to Ad-DCN transduction. However, some proliferative cells are still seen after Ad-DCN transfection, though adenoviral vectors are known to be able to transduce both non-dividing and dividing cells . Our Ad-Decorin has also been shown to be able to modulate the signaling pathways of different growth factors such as EGF by binding to its receptors , 77. ParIn this study we have examined decorin in human MBC. We have shown that MBC tissue that is a mixture of epithelial (carcinomatous) and mesenchymal (sarcomatous) elements, is negative for decorin immunoreactivity. The result indicates that the cells of MBC tissue are not able to synthesize this oncosuppressive small PG. We have also shown using 3D MBC explant cultures that decorin cDNA transduction alters the cytological features of the MBC tissue towards a less malignant phenotype. As MBCs form a rare and highly heterogeneous malignancy with poor prognosis due to fairly unsuccessful therapies currently available, novel adjuvant therapeutical approaches are required. The results of the present study favor the use of ECM macromolecules such as decorin in order to orchestrate, reengineer, reprogram, or normalize the structure and composition of the TME to achieve a less friendly environment for the cancer cells.Online Resource 1(PDF 21 kb)Online Resource 2(PDF 19 kb)


In 1884, Italian pathologists Ettore Marchiafava and Angelo Celli described intracellular micrococci in cerebrospinal fluid, and in 1887, Anton Wiechselbaum identified the meningococcus (designated as Diplococcus intracellularis meningitidis) in cerebrospinal fluid and established the connection between the organism and epidemic meningitis. Meningococcus can cause endemic cases, clusters, and epidemics of meningitis and septicemia (From the Greek pticemia .