Salmonella typhimurium senses TNF-α and show that TNF-α treatment modulates Salmonella virulent proteins , thus changing the host-bacterial interaction in intestinal epithelial cells. We investigated the expression of 23 Salmonella effectors after TNF-α exposure. We found that TNF-α treatment led to differential effector expression: effector SipA was increased by TNF-α treatment, whereas the expression levels of other effectors, including gogB and spvB, decreased in the presence of TNF-α. We verified the protein expression of Salmonella effectors AvrA and SipA by Western blots. Furthermore, we used intestinal epithelial cells as our experimental model to explore the response of human intestinal cells to TNF-α pretreated Salmonella. More bacterial invasion was found in host cells colonized with Salmonella strains pretreated with TNF-α compared to Salmonella without TNF-α treatment. TNF-α pretreated Salmonella induced higher proinflammatory JNK signalling responses compared to the Salmonella strains without TNF-α exposure. Exposure to TNF-α made Salmonella to induce more inflammatory cytokine IL-8 in intestinal epithelial cells. JNK inhibitor treatment was able to suppress the effects of TNF-pretreated-Salmonella in enhancing expressions of phosphorylated-JNK and c-jun and secretion of IL-8. Overall, our study provides new insights into Salmonella-host interactions in intestinal inflammation.Tumor necrosis factor α (TNF-α)is a host inflammatory factor. Bacteria increase TNF-α expression in a variety of human diseases including infectious diseases, inflammatory bowel diseases, and cancer. It is unknown, however, how TNF-α directly modulates bacterial protein expression during intestinal infection and chronic inflammation. In the current study, we hypothesize that  Tumor necrosis factor α(TNF-α)is a pleiotropic inflammatory cytokine with increased expression in many human diseases. These diseases include septic shock, cancer, AIDS, multiple sclerosis, diabetes, rheumatoid arthritis, and inflammatory bowel disease -6. It isBacteria can sense signal molecules secreted by their hosts. This communication mechanism between bacterium is called "quorum sensing" (QS) ,12. QS uSalmonella is a leading cause of gastrointestinal disease worldwide. Salmonella uses the type three secretion system (TTSS), a needle-like protein transport device to inject virulence proteins into eukaryotic host cells. These virulence factors, called effectors, paralyze or reprogram the eukaryotic cell to the benefit of the pathogen [Salmonella effectors display a large repertoire of biochemical activities and modulate the function of crucial host regulatory molecules[pathogen -17. The pathogen . Salmonemolecules-22.Salmonella effectors, including AvrA, SipA, SipB, Gog B, and SpVB, have been shown to manipulate a succession of key signaling transduction pathways and physiological functions of host cells [Salmonella is one of the best characterized pathogens, it remains unknown how virulence effector gene expression changes in response to host factors, such as TNF-α.Effectors are encoded via specific pathogenicity island 1 (SPI-1) and 2 (SPI-2). Over 30 st cells . AvrA, Sst cells ,23,24. Tst cells -22. Recest cells . AlthougSalmonella strains, AvrA is an acid-inducible effector that is strongly correlated with food hygiene and food-borne infection [Salmonella to grow in the intestine [Salmonella secreted factor L (SseL) [Salmonella effectors [In nfection -27. Our nfection -32. Stimntestine . Effectontestine ,34-41. UL (SseL) -44, SspHL (SseL) , SptP, affectors ,46,47.Salmonella can invade and replicate within intestinal epithelial cells during the infection process [Salmonella serotypes such as Salmonella typhimurium provoke an intense intestinal inflammatory response, consisting largely of neutrophil migration across the epithelial lining of the intestine [S. typhimurium-infected laboratory animals and cultured epithelial cells have shown that bacteria rapidly enter epithelial cells after transient degeneration of the host cell surface microvilli and induce inflammatory responses [S. typhimurium to enter epithelial cells constitutes a crucial step in pathogenesis. Salmonella invasion of the intestinal epithelium requires the virulence-associated TTSS [Salmonella invasion [Salmonella-M cell interactions, however, are not well understood. Clearly, studying effectors can uncover important mechanisms of regulation in host-bacteria interaction.Intestinal epithelial cells are physically linked by intercellular junctional complexes that regulate multiple functions including polarity, mechanical integrity, and signaling capacity . Salmone process . Nontyphntestine ,51. Studesponses -58. Not ted TTSS ,34,53,59invasion . The facSalmonella gastroenteritis increases short- and long-term risk of inflammatory bowel disease [Salmonella is potentially exposed to TNF-α. In the current study, we hypothesize that Salmonella senses the host inflammatory factor TNF-α and that TNF-α treatment modulates Salmonella TTSS effectors, thus changing the host-bacteria interaction. We investigated the gene expression of Salmonella effectors changed by TNF-α and responses of the human intestinal cells to TNF-α treated Salmonella. We verified the expression levels of some effector proteins by Western blots. Furthermore, we used human intestinal epithelial cells as our experimental model to explore bacterial invasion and the proinflammatory NF-κB and c-Jun N-terminal kinase (JNK) signaling pathways in response to Salmonella strains with or without TNF-α pre-treatment. We found that TNF-α treatment modulated effector expression in a differentiated manner. Salmonella strains pre-treated with TNF-α induced more bacteria internalization and a more severe inflammatory response in intestinal epithelial cells than untreated Salmonella strains. Our study provides new insights into host factor regulation of bacterial effector expression through inflammatory responses.A recent study demonstrated that  disease . Chronic disease . TherefoSalmonella strains , and an AvrA mutant strain lacking the AvrA gene (SL1344AvrA-)  [S. typhimurium 14028s AvrA - was generated in our laboratory based on previously published methods by Hamilton et al., and Miller et al. [Salmonella chromosome sequences, was cloned into pMAK705 (chloramphenicol resistant). The construct plasmid was transformed into the Salmonella WT14028s strain by electroporation with a Gene Pulser apparatus  and grown at 30°C on chloramphenicol plates. Resulting colonies were then grown at 42°C to select for integrants. The integrants were subsequently grown at 30°C, the temperature at which the plasmid can leave the chromosome and autonomously replicate. AvrA gene deletion was screened by PCR. AvrA deletion was also verified by Western blot using the anti-AvrA antibody. The resulting strain was named SL14028s AvrA-.um PhoPC , Salmoneversity) . Wild-tyr et al. ,65. BrieSalmonella strains were washed thoroughly with HBSS 3 times to get rid of potential TNF-α residue in the media. The bacteria were then resuspended in fresh HBSS for cell lysis or colonization in the intestinal epithelial cells.Bacteria were grown under the following conditions: non-agitated microaerophilicbacterial cultures were prepared by inoculation of 10 ml of Luria-Bertani broth with0.01 ml of a stationary phase culture with or without TNF-α (10 ng/ml), followed by overnight incubation(~18 h) at 37°C, as previously described . OvernigTotal RNA was extracted from bacteria using a Qiagen RNeasy mini kit  according to the manufacturer's protocol. Total RNA was further digested with DNase I . RNA integrity was verified by gel electrophoresis. Extracted RNA yield and purity was then determined by measuring absorbance in the 220 nm to 350 nm range. From the resulting spectra, the concentration of nucleic acids was estimated using the absorbance values at 260 nm, while the purity of each sample was determined by calculating the 260/280 and 260/230 ratios. RNA reverse transcription was performedusing a SuperScript III kit according to the manufacturer's directions. cDNA reactionproducts were then used in a quantitative PCR reaction. The reaction mixture was subjected to 29 cycles of PCR amplification using Taq polymerase . All PCR primers  and sonicated. Equal amounts of total proteins were loaded, separated by SDS-PAGE, and processed for immunoblotting with an anti-SipA antibody  or anti-AvrA antibody. For the anti-AvrA antibody, a 15-amino-acid peptide CGEEPFLPSDKADRY was designed based on AvrA amino acids 216-230. Two rabbits were injected with the peptide and a polyclonal antibody for AvrA was tested and purified, as previously described [Human colonic epithelial HCT116 cells  were grown in DMEM  supplemented with 10% (vol/vol) fetal bovine serum, 50 μg/ml streptomycin, and 50 U/ml penicillin.Infection of HCT116 cells was performed by a previously described method . BacteriS. typhimurium strain  for 30 minutes, washed, and incubated in fresh DMEM for 30 minutes as previously described [Intestinal epithelial cells were incubated with equal numbers of the indicated escribed ,68,69. CTotal RNA was extracted from epithelial cell monolayers usingTRIzol reagent . RNA integrity was verified by gel electrophoresis. RNA reverse transcription was doneusing the iScript cDNA synthesis kit according to the manufacturer's directions. The RT cDNA reactionproducts were subjected to quantitative real-time PCR usingthe MyiQ single-color real-time PCR detection system (Bio-Rad)and iQ SYBR green supermix (Bio-Rad) according to the manufacturer'sdirections. IL-8 cDNA was amplified by using primers to thehuman IL-8 gene that are complementary to regions in exon 1(5'-TGCATAAAGACATACTCCAAACCT) and overlapping the splice sitebetween exons 3 and 4 (5'-AATTCTCAGCCCTCTTCAAAAA). All expressionlevels were normalized to the GAPDH levels of the same sample, using forward (5-CTTCACCACCATGGAGAAGGC) and reverse (5'-GGCATGGACTGTGGTCATGAG)primers for GAPDH. Percent expression was calculated as theratio of the normalized value of each sample to that of thecorresponding untreated control cells. All real-time PCR reactionswere performed in triplicate. All PCR primers were designedusing Lasergene software .Salmonella-containing HBSS (1.6 × 1010 bacteria/ml) for 30 min, washed 3 times in HBSS, and incubated at 37°C for 6 hours. Cell supernatants were removed and assayed for IL-8 by ELISA in 96-well plates as described previously [HCT116 cells were cultured in DMEM, followed by incubation in eviously .Salmonella treatment. For Western blot assay, HCT116 (with SP600125 pretreatment) were incubated with Salmonella (SP600125 50 μM) 1 hour, washed three times in HBSS and incubated HBSS (SP600125 50 μM) for 1 hour, then harvested. Levels of indicated proteins were determined by Western blotting as described above. For Salmonella invasion and IL-8 ELISA: HCT116 (with SP600125 pretreatment) were incubated with Salmonella (SP600125 50 μM) 1 hour, washed three times in HBSS and incubated DMEM for 6 hours.To determine whether the effects of TNF is required for JNK, cells were treated with a JNK inhibitor SP600125 . SP600125 (50 μM) was added directly to the culture medium one hours before Data are expressed as means ± SD. All statistical tests were 2-sided. P values of less than .05 were considered to be statistically significant. Differences between two samples were analyzed by a Student's t-test. Statistical analyses were performed using SAS version 9.2 .Salmonella effectors. We used TNF-α at a concentration of 10 ng/ml, which is similar to the pathologic concentration in an inflamed intestine or patient serum [Salmonella effectors in the pathogenic Salmonella typhimurium SL1344 with or without TNF-α treatment. As shown in Fig. Salmonella effectors, those showing an upregulation of mRNA expression in response to TNF-αare shown in Fig. We first tested whether TNF-α treatment changes the mRNA expression levels of nt serum . Using RSalmonella effector AvrA inhibits the proinflammatory NF-κB pathway  and stabilizes β-catenin and IκBα [Salmonella strains and corresponding AvrA mutants with or without TNF-α treatment. SL14028s with AvrA gene expression is known to express the AvrA protein only at low pH [C is a mutation derived from SL1344 [C.Our previous studies found that the and IκBα . We reast low pH . As showm SL1344 . InteresTo confirm that TNF-α pretreatment had no effects on bacterial growth, we measured the optical density (O.D.) of the bacteria in LB after TNF-α treatment for 18 hours. Over the still culture period, no significant difference was observed between the bacterial strain SL1344 with or without TNF-α pretreatment Fig. . SimilarSalmonella strains with or without AvrA expression.In Table Salmonella to determine whether SipA protein levels respond to TNF-α treatment. As shown in Fig. Effector protein expression may be different from mRNA levels. We therefore examined strains of Salmonella with TNF-α contributes to the physiological function of Salmonella, such as invasion. To determine whether TNF-α contributed to Salmonella invasion, we counted the number of Salmonella invading the human intestinal epithelial HCT116 cells. We found that TNF-α pretreatment of Salmonella increased the amount of internalized bacteria in epithelial cells versus untreated Salmonella SL1344  pathway is known to be regulated by the Salmonella effector AvrA [Salmonella increases JNK phosphorylation [Salmonella SL1344 could enhance c-JUN, p-c-JUN, and p-JNK expression in HCT116 cells .We further hypothesized that TNF-α treatment changes tor AvrA ,71. Salmrylation . We testlls Fig. . Statistent Fig.  and 5C. F-α Fig. . In addiSalmonella increases IL-8 through both transcriptional regulation and protein expression levels [Salmonella more aggressive, inducing more severe inflammatory responses as compared to Salmonella without TNF-α treatment. We assessed the effect of TNF-α exposed Salmonella on IL-8 mRNA expression in human intestinal HCT116 cells. IL-8 mRNA real-time PCR showed that HCT116 cells significantly increased the level of IL-8 mRNA expression after TNF-α pretreated Salmonella colonization . The enhanced bacterial invasion by TNF treatment and the increased IL-8 could be two different physiological effects in the host cells. Increased bacterial invasion is not necessary to induce increased IL-8 secretion.Cytokine IL-8 expression and secretion are common readouts for inflammatory responses in the host cells . It is kn levels ,71,73,74ion Fig. . In contRNA Fig. . Both palla Fig. . Furtherion Fig. . SL1344 lla Fig. . OverallSalmonella on IL-8 secretion is through the JNK pathway, we further used the inhibitor SP600125 to treat cells, significant less IL-8 was found in the SL14028S Salmonella with SP600125 group compared to the non-inhibitor group  and the colonic epithelium by increasing bacterial adherence to the colonic mucosa [S. typhimurium, the PhoQ sensor kinase is activated by host antimicrobial peptides. PhoQ then promotes the expression of virulence genes through a phosphorelay cascade [Salmonella senses TNF-α, thus changing the expression of the bacterial effectors. Studies in Pseudomonas raise the possibility that TNF-α sensors or receptors for TNF-α are encoded for in the prokaryotic genome. Further studies on the Salmonella quorum sensing system and effector regulation will provide insights into this powerful and effective bacteria-host interaction.Our study demonstrates that henotype . Also inignaling . Norepinc mucosa . EHEC usc mucosa . The QS c mucosa . Given tc mucosa . In S. t cascade . HoweverSalmonella effector SipA. SipA contributes significantly to Salmonella host cell invasion in vitro and to Salmonella enterocolitis in vivo [Salmonella had more internalized Salmonella. Moreover, Salmonella strains with TNF-α pretreatment induced higher proinflammatory responses, such as the activation of JNK and elevation of IL-8, compared to the Salmonella strains without TNF-α exposure. This observation is correlated with the enhanced expression of SipA in Salmonella exposed to TNF-α.Our data demonstrate that TNF-α exposure increases the expression of the  in vivo ,41,79. SSalmonella SL14028s is known to be deficient in AvrA expression. AvrA protein expression was only detectable when SL14028s was cultured in low pH media [Salmonella effectors. Although AvrA is known to regulate diverse bacteria-host interactions [Salmonella effectors in response to the inflammatory status of the host cells remains unknown. Further investigations on the interaction of AvrA and its fellow effectors will help us to understand the network of Salmonella effectors in epithelial cell-bacteria cross-talk.At the mRNA level SL1344 expressing AvrA has a significant ability to modify SipA in response to TNF-α, whereas SL14028s is less responsive Fig.. We alsopH media -27. The ractions ,71,80, tin vivo model.TNF-α exposure decreased the mRNA expression of SPI-2 effectors Gog B and SpVB, which are known to promote bacterial replication and systemic spread ,20,22. WSalmonella effectors, such as SipA, gogB, and spvB. Salmonella exposed to TNF-α induced more bacterial internalization, higher activity of JNK pathway with enhanced p-JNK and p-c-Jun in the host cells. As a consequence of the activation of the JNK pathway, the expression of inflammatory cytokines, such as IL-8, is higher in cells colonized with TNF-α pretreated Salmonella. Overall, Salmonella exposed to TNF-α caused enhanced inflammation in intestinal epithelial cells. We postulate that chronic inflammation with elevated TNF-α in host cells may change the behavior of pathogenic bacterial effectors and may make pathogens more virulent.In summary, our current study answers the fundamental question of whether TNF-α expressed from host cells can change the expression level of Salmonella. Enhanced bacteria internalization and more severe inflammatory responses of intestinal epithelial cells were found after Salmonella strains were exposed to TNF-α. Activation of the JNK pathway significantly elevates and enhances inflammation in intestinal epithelial cells. As a consequence, the expression of inflammatory cytokines, such as IL-8, is high in cells colonized with TNF-α pretreated Salmonella. Our studies provide new insights into host factor TNF-α regulation of Salmonella effector expression in bacterial invasion and inflammatory responses.We found that TNF-α treatment modulated effector expression in a differential manner. The expression of effector SipA was increased after TNF-α exposure in pathogenic The authors declare that they have no competing interests.All authors read and approved the final manuscript.JM: Participated in the experimental design, preparation of the RNA sample, PCR analysis, Western Blots, ELISA, acquisition of data, analysis and interpretation of data, and drafting of tables and figures. JM also helped to write Methods for the manuscript.YZ: Participated in growing bacteria, bacterial invasion assays, Western Blots, ELISA, JNK inhibitor treatment, AvrA mutant strain establishment, interpretation of the data, statistical analysis, analysis and interpretation of data, and drafting of tables and figures.YX: Performed statistical analysis and made critical revision of the manuscript for intellectual content.JS: Participated in the study concept and design, acquisition of data, analysis and interpretation of data, material support, drafting and critical revision of the manuscript for important intellectual content. JS also obtained funding and supervised the study.


TNF-α) gene polymorphism G-308A and total serum immunoglobulin E (TsIgE) levels in the onset of pseudoexfoliation glaucoma (PEXG) in Pakistani patients.The purpose of the present study was to determine the role of the tumor necrosis factor alpha  and restriction fragment length polymorphism (RFLP). TsIgE levels were determined by solid-phase enzyme-linked immunosorbent assay (ELISA).The The AA and GA genotypes were strongly associated with PEXG (p<0.001), with an odds ratio (OR) of 0.07  and 0.24 (95% CI=0.12-0.51), respectively, while the GG genotype was found at a higher frequency in controls as compared to patients (p<0.001) OR=8.95 (95% CI=4.55–17.81). No significant difference was found in TsIgE levels of both patients and controls (p=0.86).TNF-α polymorphism G-308A is strongly associated with PEXG. To our knowledge this is the first study in southeast Asia which demonstrates a strong association of a TNF-α polymorphism with PEXG.The present study concludes that the  Pseudoexfoliation syndrome (PEX) was first described by Lindberg in 1917 and involves excessive production and progressive accumulation of fibrillar extracellular material in various tissues . AlthougPEX syndrome occurs worldwide but its prevalence varies among different age groups, races, and geographical locations. The highest prevalence of 20–70% has been reported in elderly people 70–80 years) and it is estimated that PEXG affects 10– 20% of all individuals above 60 years of age. Moreover, prevalence varies in different countries with the highest rate of about 46.9% in Greece . Compreh0 years aTNF-α or TNF-α receptor-1 (TNF-R1) are raised in the retina of glaucomatous eyes as compared to normal eyes, and therefore it was suggested that cell death mediated by TNF-α is a contributing factor in the neurodegeneration in glaucoma ). The distribution of allele frequencies (2 74.6).The GG wild-type genotype was found at a frequency of 87% in the controls and 43% in the PEXG patients with an odds ratio (OR) of 8.95 . In addition, the variant A allele was found to be more prevalent in the PEXG male patients as well as female patients when compared to the appropriate controls . The frequency of the A allele was found to be nearly the same in both sexes  of patients and controls were compared , which in turn mediates the expression of a wide range of genes essential for neuronal survival. Contrary to its neuroprotective role, TNF-α can also serve as a neurodegenerative factor when it binds to the low affinity death receptor TNF-R1 and induces the mitochondria-mediated apoptotic pathway –29. ThusTNF-α polymorphism G-308A with PEXG in both Turkish and Caucasian populations [TNF-α as compared to normal basal level transcription, as a result of which increased levels of TNF were observed. Recently, a significant association has been observed for the G-308A polymorphism between PEXG patients and controls in the Iranian population (p<0.005) [In the present study we found a strong association of GA and AA genotypes with PEXG, suggesting a role of the A allele in the pathogenesis of the disease. However, our results are in contradiction with previous studies, which have shown no significant association of the ulations ,33. Agarulations  reportedp<0.005) , which ip<0.005) , which ip<0.005)  in the Ip<0.005) .Our results are in accordance with the findings of Razenghinejad et al. , and areTNF-α is essential to circumvent the deleterious effects of overexpression by transcriptional upregulation. Thus excessive production of TNF associated with the G-308A polymorphism may have an important role in the development of TNF2-associated autoimmune diseases, and may act as a genetic susceptibility factor driven by a high TNF-α expression, which would subsequently lead to immune responses causing the onset of different diseases. A recent study by Sawada et al. [A strong association of this polymorphism has also been observed in the Chinese population, but that study was performed in patients with primary open-angle glaucoma (POAG) and with a very small sample size . Althouga et al.  also demIt is worth mentioning here that the greater number of carriers with a GA genotype in the PEXG patients also points toward the possibility of a dominant mode of inheritance of the genetic defects responsible for PEXG. However, due to the presence of such carriers in normal individuals, although at much lower frequency, it is likely that environmental influences and other genetic factors contribute to the development of the disease.On the basis of different evidence it has been suggested that autoimmune damage to the optic nerve may occur directly by autoantibodies or indirectly by a “mimicked” autoimmune response to a sensitizing antigen, which in turn injures retinal ganglion cells . In a reTNF-α polymorphism G-308A to be strongly associated with the pathogenesis of PEXG, and thus propose that the GA and AA genotypes of the TNF-α regulatory region can be considered genetic markers for the stratification of PEXG patients in Pakistan. Mutations in TNF-α might be involved in causing different neurodegenerative disorders, therefore it is important to consider it as a novel therapeutic target for the treatment of neurodegenerative diseases like glaucoma. We expect that our study will prove to be a helpful tool in the treatment and management of PEXG by opening up new horizons in the field of target discovery to develop novel therapeutics.In conclusion, we find the A allele of the


TNF-α induced endothelin-1 gene expression was associated with a parallel increase in the release of the corresponding peptide in the culture medium. These findings suggest that the enhanced synthesis and release of endothelin-1 occurring in conditions of increased generation of TNF, may act as a modulatory factor that counteracts the hypotensive effect and the excessive platelet aggregation and adhesion induced by TNF.We have studied the effect of human recombinant tumour necrosis factor-α (TNF-α) on gene expression and production of endothelin-1 in cultured bovine aortic endothelial cells. TNF-α (10 and 100 ng ml


Previous studies led to the hypothesis that the use of TNF-α in isolated limb perfusion causes specific destruction of tumour endothelial cells and thereby induces an increased permeability of tumour vasculature. However, whether TNF-α contributes to the therapeutic efficacy in IHP still remains unclear. In an in vivo rat liver metastases model we studied three different tumours: colon carcinoma CC531, ROS-1 osteosarcoma and BN-175 soft-tissue sarcoma which exhibit different degrees of vascularisation. IHP was performed with melphalan with or without the addition of TNF-α. IHP with melphalan alone resulted, in all tumour types, in a decreased growth rate. However in the BN-175 tumour addition of TNF-α resulted in a strong synergistic effect. In the majority of the BN-175 tumour-bearing rats, a complete response was achieved. In vitro cytoxicity studies showed no sensitivity (CC531 and BN-175) or only minor sensitivity (ROS-1) to TNF-α, ruling out a direct interaction of TNF-α with tumour cells. The response rate in BN-175 tumour-bearing rats when TNF-α was coadministrated with melphalan was strongly correlated with drug accumulation in tumour tissue, as only in these rats a five-fold increased melphalan concentration was observed. Secondly, immunohistochemical analysis of microvascular density (MVD) of the tumour showed a significantly higher MVD for BN-175 tumour compared to CC531 and ROS-1. These results indicate a direct relation between vascularity of the tumour and TNF-α mediated effects. Assessment of the tumour vasculature of liver metastases would be a way of establishing an indication for the utility of TNF-α in this setting.Isolated hepatic perfusion (IHP) with melphalan with or without tumour necrosis factor alpha (TNF- Here we present data that indicate that the antitumour effect of TNF-α is correlated with the tumour microvessel density.Based on our findings in the ILP studies, it is indicated to study whether TNF-We used male inbred WAG/RIJ or Brown-Norway (BN) strain rats, weighing 250–300 g, obtained from Harlan-CPB . The rats were fed a standard laboratory diet. All animals were housed under standard conditions of light and accommodation. The protocol was approved by the committee for animal research of the Erasmus University, Rotterdam, The Netherlands. The experimental protocols adhered to the rules outlined in the Dutch Animal Experimentation Act of 1977 and the published Guidelines of the UKCCCR for the Welfare of Animals in Experimental Neoplasia .in vivo is about 6–8 days. The spontaneously originated nonimmunogenic osteosarcoma ROS-1 is also transplantable in the WAG-RIJ rat and in the liver metastases model it has a mean doubling time of about 4–5 days. The spontaneously originated nonimmunogenic soft-tissue sarcoma BN-175 is the fastest growing tumour of the tumours tested, with an estimated doubling time in vivo of about 2–3 days and is transplantable in syngeneic BN rats. Following a standardised protocol, small viable tumour fragments of CC531, ROS-1 or BN-175 tumour fragments of 1×2 mm2 were implanted under the liver capsule, one on the left and one on the right side of the left liver lobe, using a 19 G Luerlock needle. Experiments started at a fixed tumour diameter between 5 and 6 mm. When tumours reached a size of 20 mm in diameter or animals showed obvious signs of discomfort the animals were killed.Three different tumours were used in this study. The weakly immunogenic colon carcinoma CC531 is a 1,2-dimethylhydrazine-induced, moderately differentiated adenocarcinoma transplantable in syngeneic WAG/RIJ rats. The estimated doubling α (4.9–5.8×107 U mg−1) was provided as a kind gift by Boehringer Ingelheim GmbH, Ingelheim/Rhein, Germany. Melphalan  was obtained as a sterile powder (100 mg) that was dissolved aseptically using solvent and diluent provided by Burroughs Wellcome .Recombinant human TNF-This rat isolated liver perfusion model has been described in detail earlier by Figure 1−1 was maintained with a low-flow roller pump . Rats were perfused for ten min with oxygenated Haemaccel in which melphalan and/or TNF-α was dissolved. This short perfusion time was used as we observed rapid clearance of melphalan from the perfusate in this time frame. Secondly, perfusion of the liver beyond 10 min may increase the risk for tissue damage to the liver, but also to the gut as blood flow to the gut is impaired during the perfusion. Afterwards a washout was performed by perfusing with 10 ml of oxygenated Haemaccel. Heparin (50 IU)  was added to the perfusate. The perfusate was oxygenated in a reservoir with a mixture of O2/CO2 (95% : 5%) and was kept at 38–39°C by means of a heat exchanger and a warm water bath. A temperature probe was positioned in the lumen of the arterial catheter, 5 cm from the catheter tip.Isolation of the hepatic vascular bed was obtained by temporarily ligating the common hepatic artery and the portal vein. The venous outflow limb was isolated by temporarily clamping the supra-hepatic caval vein and by applying a temporary ligature around the infra-hepatic caval vein containing the cannule, cranial to the right adrenal vein. The mesenteric artery was temporarily clamped in order to reduce splanchnic blood pressure. The circuit was primed with 10 ml Haemaccel . Arterial flow of 5 ml minFollowing the washout procedure, the clamps on caval vein, portal vein, hepatic artery and mesenteric artery were released. The gastroduodenal artery and femoral vein were ligated and the gastroduodenal and femoral cannulas were removed.partial tumour response was chosen for this study. So in the case of additive or synergistic effect of TNF-α on melphalan this could still be demonstrated in the growth curves of the tumours. All animals underwent IHP only once. CC531-bearing rats were treated with 50 μg melphalan (n=6), 20 μg TNF-α (n=6) or a combination of 50 μg melphalan and 20 μg TNF-α (n=6). ROS-1-bearing rats were perfused with 50 μg melphalan (n=6), 20 μg TNF-α (n=8), or a combination of 50 μg melphalan and 20 μg TNF-α (n=6). In the BN-175-bearing rats perfusions were carried out with 200 μg melphalan (n=6), 20 μg TNF-α (n=6), or a combination of 200 μg melphalan and 20 μg TNF-α (n=6). After IHP tumour size was measured via a small midline laparotomy every fourth day. Tumour volume was calculated by using the following formula: tumour volume=A2×B×0.4, where B is the largest diameter and A the diameter perpendicular to B, measured with a standardised calliper. In every treatment group, sham perfused rats (n=6) and untreated control rats (n=5) were included.Treatment started at a fixed tumour size of 5–6 mm in diameter. Rats were perfused in random order. In a pilot dose finding study performed for each tumour type the melphalan dose inflicting a −1), 1% streptomycin (5000 IU ml−1) and 1% L-glutamine (200 mM)  in a humidified incubator at 37°C and 5% CO2. Before usage, the cells were trypsinised , centrifuged , resuspended and the viability measured by trypan blue exclusion. For in vitro testing of proliferation inhibition, 1.0×104 viable cells were seeded in flat-bottomed 96-well microtiter plates . After 24 h the cells were incubated with different concentrations of TNF-α for 72 h ranging from 0 to 10 μg ml−1. Afterwards, cells were washed with PBS and fixed for 1 h with 10% trichloroacetic acid at 4°C. Growth of tumour cells was measured using the sulpharhodamine-B assay according to the method of CC531 and BN-175 cells were grown in RPMI 1640 and ROS-1 cells in modified Eagle's medium  supplemented with 10% foetal calf serum , 1% penicillin . p-[Bis(2-chloroethyl)amino]-phenylacetic acid methyl ester was used as an internal standard. Samples were extracted over trifunctional C18 silica columns. After elution with methanol and evaporation, the compounds were derivatised with trifluoroacetic anhydride and diazomethane in ether. The stable derivates were separated on a methyl phenyl siloxane GC capillary column and measured selectively by single-ion monitoring GC–MS in the positive EI mode described earlier by After 5 min of the restoration of the circulation, the perfused tumour and part of the liver were excised. The tissues were immediately frozen in liquid nitrogen to stop metabolism of melphalan and stored at –80°C. Tumour and liver tissues were homogenised in 2 ml acetonitrile  and centrifuged at 2500 Cryosections of tumours were fixed for 15 min with 4% formaldehyde. After rinsing with PBS, sections were incubated for 1 h with 1 : 10 PBS diluted, mouse-anti-rat-endothelial cell antibody . For the negative control an aspecific mouse IgG was used . Thereafter, sections were rinsed with PBS and incubated for 1 h with 1 : 100 diluted, in 5% normal rat serum in PBS, goat-anti-mouse peroxidase-labelled antibody . After rinsing with PBS, positive cells were revealed by immunoperoxidase reaction with DAB solution  and counterstained with haematoxylin. For microvessel quantification two independent persons performed a blinded analysis. Positive cells were counted in three different high-power fields (magnification ×160) in each slide according to the method of In vitro bioassays and in vivo tumour response results were evaluated for statistical significance with the Mann–Whitney U-tests with SPSS8.0 for Windows 98. Mann–Whitney U-test was used to compare melphalan concentrations in different groups and Kruskal–Wallis test to compare number of positive cells in different tumours. A significance level of P<0.05 was used in all analyses.α was evaluated for the CC531, ROS-1 and BN-175 tumour starting at an equal size of 5–6 mm in diameter. In all groups, sham IHPs with only perfusion medium were performed. The graphs in Figure 2α, a dramatically enhanced tumour response was observed in all animals. This is a significant reduction of mean tumour volume compared with rats perfused with either TNF-α only or melphalan alone . In the CC531 or ROS-1 tumours, synergy between TNF-α and melphalan was not observed.The antitumour efficacy of IHP with melphalan with or without TNF-α on the growth of tumour cells in vitro was determined to evaluate whether the synergistic effect of TNF-α could be related to direct tumour cell toxicity. The calculated concentration of TNF-α in the perfusate during IHP in vivo is about 1.5 μg ml−1. So in vitro tumour cells were exposed to a range of TNF-α concentrations varying from 0 to 10 μg ml−1. The growth curves are shown in Figure 3α, a growth inhibition of up to 30% at 10 μg ml−1 was observed.The effect of TNF-α is 20% of the dose used in ILP, an enhanced drug accumulation in tumour tissue might take place as well, as observed after TNF-α based ILP. In order to investigate this mechanism, melphalan concentrations were measured in tumour and liver tissues after IHP with melphalan with and without TNF-α. In the CC531 and ROS-1, tumours, melphalan concentration did not increase significantly after IHP with melphalan and TNF-α . So an augmented drug accumulation can also be achieved in the IHP setting when TNF-α is coadministered.In this perfusion setting, in which the dose of TNF-as equal . After Iα by increasing leakage of tumour vessels enhances intratumoural concentrations of chemotherapeutics. The increased uptake of melphalan might therefore be correlated with the microvessel density (MVD) of the tumour. Quantification of the MVD was performed by immunohistochemical staining of endothelial cells. The microvessel count of the colon carcinoma CC531 and the osteosarcoma ROS-1 was equal  or bigger (7–8 mm) tumours comparable tumour responses were observed (data not shown). We are of the opinion that this observation is essential in understanding and explaining the impressive responses demonstrated.In the present study, we demonstrated that addition of TNF-α was studied by Alexander et al, (1998). Vascular permeability was measured by diffusion of radiolabelled 131I albumin in liver and tumour tissue. A significant increase of the 131I albumin postperfusion could be demonstrated compared to levels 131I albumin measured before perfusion. However, this rise was equal in tumours perfused with or without TNF-α. A TNF-α independent mechanism of the increased endothelial permeability was suggested by the authors. However, in the present study, we demonstrated that TNF-α is effective in increasing vascular permeability for melphalan selectively in tumour tissue. A more important finding, however, is that this effect could only be found in the highly vascularised BN-175 tumour. The results of Alexander et al, reported on intratumoural 131I albumin concentrations were mainly based on colorectal carcinoma liver metastases. In hypovascular rat colon carcinoma, we also could not find an increase of melphalan intratumourly. We therefore hypothesize that the usual hypovascularity of colorectal metastases in patients explains the lack of TNF-benefit in the experience as described by Alexander in patients, which correlates closely to our observations in our hypovascular colon cancer liver metastases model in rats.Changes in vascular permeability in patients who underwent IHP with TNF-α performed in patients with metastases of ocular melanoma or leiomyosarcoma showed overall response rates of 50–52% (αvs 6 months after IHP without TNF-α (α in patients with colorectal liver metastases the mean duration of response was in both groups 8–10 months (α has therapeutic potential in IHP. In patients with colorectal liver metastases however, IHP with melphalan alone may well be just as effective as combined with TNF-α. Assessment of the degree of tumour vasculature of liver metastases would be a way of establishing an indication for the utility of TNF-α in this setting.IHP with melphalan and TNF-


In vitro and in vivo studies have suggested synergistic anti-tumour activity of combined hyperthermia and tumour necrosis factor alpha . However, some studies indicated an increased systemic toxicity of TNF by additional hyperthermia. The aim of this study was to obtain starting dosages for a clinical phase I study on the application of deep local hyperthermia and systemic TNF. We investigated the effect of local hyperthermia on the toxicity and efficacy of systemic TNF. Rats (Wag/Rij) carrying a subcutaneously transplanted osteosarcoma in the hind leg received a single intravenous dose of recombinant human (rh) TNF-alpha, either at normothermia or at hyperthermia, by positioning the tumour bearing hind leg in a water bath of 43 degrees C. Dose-effect curves for lethality and tumour cure were established and LD50 and TCD50 values were calculated. Systemic toxicity was increased by local hyperthermia. The LD50 values (+/- s.e.) were 1088 (+/- 61) micrograms kg-1 at normothermia and 205 (+/- 23) micrograms kg-1 at hyperthermia, resulting in a thermal enhancement ratio (TER) of 5.3. Following normothermia, tumour cures were observed at TNF concentrations of 1000-1300 micrograms kg-1, while this was observed at doses of 50-300 micrograms kg-1 when combined with hyperthermia , resulting in a TER of 6.4. Systemic toxicity and anti-tumour activity of TNF are both increased by local hyperthermia. A safe starting dose for the combined clinical treatment would be 10% of the dose of TNF-alpha that has been recommended for phase II studies on intravenous bolus administration of TNF-alpha at normothermia. In view of the large variability in tumour sensitivity for TNF-alpha, the clinical usefulness of this combined treatment modality has to be determined.


Niemann-Pick type C (NPC) disease is a lysosomal storage disease characterized by the accumulation of cholesterol and glycosphingolipids. The majority of NPC patients die in their teen years due to progressive neurodegeneration; however, half of NPC patients also suffer from cholestasis, prolonged jaundice, and hepatosplenomegaly. We previously showed that a key mediator of NPC liver disease is tumor necrosis factor (TNF) α, which is involved in both proinflammatory and apoptotic signaling cascades. In this study, we tested the hypothesis that blocking TNF action with an anti-TNF monoclonal antibody (CNTO5048) will slow the progression of NPC liver disease.Treatment of wild-type C57BL/6 mice with NPC1-specific antisense oligonucleotides led to knockdown of NPC1 protein expression in the liver. This caused classical symptoms of NPC liver disease, including hepatic cholesterol accumulation, hepatomegaly, elevated serum liver enzymes, and lipid laden macrophage accumulation. In addition, there was a significant increase in the number of apoptotic cells and a proliferation of stellate cells. Concurrent treatment of NPC1 knockdown mice with anti-TNF had no effect on the primary lipid storage or accumulation of lipid-laden macrophages. However, anti-TNF treatment slightly blunted the increase in hepatic apoptosis and stellate cell activation that was seen with NPC1 knockdown.Current therapeutic options for NPC disease are limited. Our results provide proof of principle that pharmacologically blocking the TNF-α inflammatory cascade can slightly reduce certain markers of NPC disease. Small molecule inhibitors of TNF that penetrate tissues and cross the blood-brain barrier may prove even more beneficial. NPC1 gene Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal storage disease characterized by the accumulation of cholesterol and glycosphingolipids. Ninety-five percent of clinical cases are caused by mutations in the We previously showed that tumor necrosis factor (TNF) α is a key mediator of NPC liver disease In this study, we have tested the hypothesis that blocking TNF-α action with an anti-TNF-α monoclonal antibody (CNTO5048) will slow the progression of NPC liver disease. Targeting TNF-α mediated inflammation is not expected to halt the primary lysosomal lipid accumulation, but it may reduce secondary consequences of NPC liver disease. Our results indicate that anti-TNF treatment has only a modest effect in blunting the hepatic apoptosis and stellate cell activation that is characteristic of NPC disease.To ensure that anti-TNF is efficacious in our model system, we treated wild-type mice for 7 days with either anti-TNF or saline. We then challenged the mice with injection of either lipopolysaccharide (LPS) or saline. The pathological effects of LPS are, in part, mediated by the release of TNF-α Control mice treated with saline injections and no LPS challenge had undetectable plasma levels of IL-6 (<4 pg/ml). Two mice treated with saline injections and then subjected to an LPS challenge had 68 and 74 ng/ml of plasma IL-6. Two mice treated with anti-TNF injections and then subjected to an LPS challenge had reduced levels of plasma IL-6, at 27 and 29 ng/ml.Hepatic NPC1 knockdown in mice caused TNF-α-mediated hepatic inflammation that was markedly less severe than that seen with LPS treatment. We injected three mice with NPC1-specific antisense oligonucleotides (ASOs) twice a week for 15 weeks to induce NPC liver disease Our experimental protocol had twenty C57BL/6 mice divided into four treatment groups. Ten mice each were injected twice a week with NPC1-specific ASOs or with mismatched control ASOs. Five mice in each group were injected once a week with saline or with the anti-TNF-α monoclonal antibody. NPC1 protein levels in the liver were knocked down to less than 10% of control levels by NPC1 ASO treatment . Anti-TNPreviously, we showed that TNF-α does not play a role in the development of the primary storage phenotype of NPC Serum alanine aminotransferase (ALT) activity was measured in the four treatment groups to determine whether anti-TNF treatment of NPC1 knockdown mice resulted in less liver injury. Mice injected with mismatched ASOs and treated with saline or anti-TNF had similar serum ALT activities of 23.6±4.5 and 25.2±5.4 U/L, respectively. NPC1 knockdown led to a significant increase in serum ALT to 98±12.9 U/L. The serum ALT activity of NPC1 knockdown mice was not significantly reduced by anti-TNF treatment (78.3±20.7 U/L). These data indicate that anti-TNF has no effect on the hepatic injury that leads to enzyme release.Sections of mismatched, control ASO-treated livers showed few foamy, vacuolated macrophages seen in . Saline Thus far there was only one indication that anti-TNF has a therapeutic effect on the NPC knockdown mice. That is that the liver weight did not increase significantly when NPC was knocked down in anti-TNF-treated mice. Next we examined the downstream consequences of hepatic lipid accumulation.Knockdown of NPC1 was shown previously to result in a 10-fold increase in the number of apoptotic hepatocytes, which was offset by proliferation of hepatic stellate cells NPC1 knockdown also results in increased proliferation of hepatic stellate cells, which can be visualized by α-smooth muscle actin immunohistochemistry . QuantifThus, the second and third indications that anti-TNF had a therapeutic effect were that the numbers of apoptotic cells and proliferating stellate cells did not increase significantly when NPC1 was knocked down in anti-TNF treated mice.The goal of the current study was to evaluate an anti-TNF-α monoclonal antibody as a potential therapeutic modality for NPC disease. Our previous results had shown that TNF-α mediates some of the secondary consequences of NPC disease in the liver. NPC1 knockdown mice showed hepatomegaly, liver cholesterol accumulation, elevated serum liver enzymes, increased hepatocyte apoptosis and activation of stellate cells Our results show that, as expected, anti-TNF had no effect on the primary NPC phenotype. NPC1 knockdown mice treated with vehicle or anti-TNF showed the same hepatic cholesterol storage, number of foamy cells throughout the hepatic parenchyma and release of liver enzymes into the blood. Anti-TNF treatment of NPC1 knockdown mice led to fewer apoptotic hepatocytes and activated stellate cells than did vehicle treatment; however, the attenuation did not reach statistical significance. Instead, anti-TNF treatment led to a phenotype that was intermediate between control and NPC1 knockdown.NPC is a lysosomal storage disease with a primary impairment in intracellular cholesterol trafficking Here we tested whether anti-TNF treatment would be efficacious for NPC patients. The monoclonal antibody that we used in our study binds and neutralizes murine TNF-α; it was a surrogate for infliximab, a monoclonal antibody that binds the membrane-bound and soluble forms of human TNF-α and prevents their binding to cellular receptors 5′CCCGATTGAGCTCATCTTCG3′. As a control, we used an ASO with a mismatched sequence 5′CCTTCCCTGAAGGTTCCTCC3′. ASOs were dissolved in 0.9% saline and stored at −20°C.The 20-mer 2′-0-methoxyethyl modified ASOs were synthesized and purified as described previously Guide for the Care and Use of Laboratory Animals.Ethics Statement: All of the following procedures were approved by the Institutional Animal Care and Use Committee at Tufts University  and were in compliance with the NIH Female C57BL/6 mice (4 wks of age) were purchased from The Jackson Laboratory . They were housed 5 animals per cage and fed rodent chow. Mice were challenged with LPS by intraperitoneal injection of 200 µl of a 1-mg/ml solution (Sigma L5293). Mice were injected intraperitoneally with either NPC1 ASO or a mismatched control ASO at a dose of 100 mg/kg/wk for 9 weeks. They were also injected intraperitoneally with either saline or an anti-TNF monoclonal antibody (CNTO5048) at a dose of 12 mg/kg/wk for 9 weeks. At the end of the treatment period, animals were fasted overnight, then euthanized and blood samples taken by cardiac puncture. Mice were perfused with cold PBS via cardiac puncture, after which tissues were dissected and fixed in 10% formalin or snap-frozen in liquid nitrogen.Liver tissue was blotted for NPC1 as previously described Aliquots of 400 µg liver homogenate were subjected to Folch extraction g for 30 minutes. Serum was sent to IDEXX Laboratories  for analysis of liver enzymes.Blood was allowed to clot for 30 min, then subjected to centrifugation at 1000× Liver tissue was fixed in 10% formalin and paraffin embedded. Four-micron sections were cut and stained with Masson Trichrome, hematoxylin-eosin or anti-α smooth muscle actin by the Tufts Animal Histology Core or the Tufts Medical Center Histology Department. Slides were viewed using a Zeiss Axioplan microscope with a 20X objective. The number of hepatocytes and foamy macrophages were quantified per field. α-Smooth muscle actin immunohistochemistry was quantified using ImageJ. For all analyses, 10 random fields were examined per liver; 3–5 animals were evaluated per treatment group.Apoptotic cells in formalin-fixed, paraffin-embedded liver tissue sections were detected using the DeadEnd Fluorometric TUNEL System  according to the manufacturer's protocol. The number of apoptotic cells was quantified per field. Ten random fields were examined per liver; 5 animals were evaluated per treatment group.All values are expressed as means ± SD. Statistical analysis was performed by one way ANOVA followed by Tukey's honestly significant differences test using the Vassar Stats website.


A prospective study was performed to assess the use of plasma measurement of tumour necrosis factor (TNF), lymphotoxin alpha  and their soluble receptors (p55 and p75) for prognostic risk assignment in 61 patients with Hodgkin's disease. Plasma levels of TNF, p55 and p75, but not of LT alpha, were higher in Hodgkin's disease patients than in healthy controls. Plasma levels of TNF, p55 and p75 were associated with several prognostic factors for Hodgkin's disease, including those related to the host  and to the tumour . Elevated plasma levels of TNF, p55 and p75 were also associated with several parameters reflecting an immune activation, including the presence of B symptoms, elevated serum levels of gammaglobulins, alkaline phosphatase and fibrinogen, as well as peripheral monocytosis, anaemia and low serum albumin levels. Finally, elevated TNF ligand receptor plasma markers were associated with a lower incidence of complete response to therapy and predicted shorter free-from-progression survival and overall survival of the patients. These results indicate that the plasma levels of TNF and its soluble receptors correlate with clinical features and outcome of patients with Hodgkin's disease.


P=0.0012). In addition, TNF−859T was found in 26.0% of patients compared with 14.4% of the controls (P=0.0036). The two loci were in tight linkage disequilibrium, that is, almost all the individuals having TNF+488A also had TNF−859T. Patients with the TNF+488A or TNF−859T were more likely to present with a moderately differentiated tumour than those patients without the uncommon allele. In all, 16.7% of patients with TNF+488A and 29.9% of patients without TNF+488A presented with a G1 tumour (P=0.015). A total of 14% of patients with TNF−859T and 30.5% of patients without TNF−859T presented with a G1 tumour (P=0.0043). There was no significant effect on time to first recurrence, stage progression or grade progression. In conclusion, a significant association between TNF polymorphisms TNF+488A and TNF−859T and risk of bladder cancer was detected in this study. Both these polymorphisms were associated with grade of tumour at presentation although there was no significant effect on subsequent tumour behaviour.The purpose of this study is to assess the role of tumour necrosis factor (TNF) polymorphisms in the risk of developing bladder cancer and effect on tumour stage, grade and progression. In all, seven single-nucleotide polymorphisms in TNF were studied in 196 bladder cancer patients and 208 controls using a PCR-SSP genotyping technique. It was seen that there was a significant association of two polymorphisms in TNF with bladder cancer: the TNF+488A allele was found in 28.1% of patients compared with 14.9% of controls ( Transitional cell carcinoma (TCC) comprises 90% of all cases of bladder cancer. An estimated 57 400 new cases (42 000 men and 15 200 women) will be diagnosed with TCC in the USA in 2003. A total of 12 500 people (8600 men and 3900 women) will die of the disease .α) has been implicated in the development and progression of tumours  may predispose to risk of bladder cancer or alter subsequent tumour behaviour.There is considerable direct and indirect evidence to implicate TNFα is found on chromosome 6 in the class III region of the major histocompatibility complex (MHC) and is flanked by the lymphotoxin α and β genes. There are 16 known polymorphisms in and around TNF, including five microsatellites. The two most studied single-nucleotide polymorphisms (SNPs) are the promoter polymorphisms −308G/A and −238G/A. Other polymorphisms that may be of significance are +488G/A in the first intron, and the promoter variants, −1032T/C, −865C/A, −859C/T and −380G/A. There have been many efforts to determine the significance of these polymorphisms with extensive disease association and functional studies, the results of which have been conflicting  genotyping technique was performed using protocols previously described .P=0.0068) and in the second set, in 16.8% of controls compared with 27.8% of patients  and in the second set, in 15.8% of controls compared with 25.8% of patients  and TNF+488A  and there were also significantly more patients with G2+G3 vs G1 in the TNF−859T polymorphism group (P=0.023).Information on grade of tumour at presentation was available on 191 patients. There was a significant association between the grade of tumour at initial presentation and the polymorphisms TNF−859T . When G2 and G3 were considered together, that is, G1 vs G2+G3, the association approached significance (P=0.06), see Of the 54 patients with the TNF+488A polymorphism, nine (16.7%) presented with a G1 tumour. Of the 137 patients who did not carry this variant, 41 (29.9%) presented with a G1 tumour. Again, No association was found with polymorphisms and stage at presentation.No association was found with polymorphisms and number of lesions at presentation.P=0.57, TNF−859T, P=0.41), time to first stage progression  or on time to first grade progression . The median time of follow-up from first diagnosis was 3.65 years. The range was 0.06–35.8 years.The log-rank test showed no association of TNF+488A or TNF−859T on time to first recurrence (TNF+488A, The role of TNF polymorphisms in the risk of bladder cancer, bladder cancer phenotype and subsequent tumour behaviour was assessed in this study. We have shown for the first time a significant association of the TNF polymorphisms TNF+488A and TNF−859T and risk of bladder cancer. The TNF+488A has been previously associated with rheumatoid arthritis  and commα production despite demonstrating a clear association with rheumatoid arthritis, suggesting that the disease associations are the result of linkage disequilibrium with alleles within or near the TNF/Lymphotoxin locus.In addition to the association of TNF+488A and TNF−859T with risk of bladder cancer, we have shown for the first time a very strong linkage disequilibrium between these two sites in bladder cancer patients, normal controls and melanoma patients. The linkage disequilibrium between TNF+488A and TNF−859T demonstrates the presence of a previously unreported haplotype; one that we hypothesise will have functional significance. Several TNF haplotypes have already been described involving TNF and the surrounding lymphotoxin gene (The mechanisms that could relate polymorphisms that upregulate inflammatory pathways to cancer are well recognised , includiα or TNFα-dependent pathways direct the progression of bladder cancer to grade 2, but other additional factors are involved in progression from grade 2 to grade 3, in keeping with the evidence of stepwise progression of bladder cancer. For example, in other types of cancer, clear upregulation of oncogenes to cause a specific subtype of disease are recognised egg AML and ALL.In addition to their role in the risk of bladder cancer, we investigated the role of TNF polymorphisms in tumour behaviour. We have shown a significant association of both TNF+488A and TNF−859T with grade of tumour at presentation. If the presence of TNF+488A or TNF−859T affects the development of bladder tumours to make them generally less well differentiated, then we would expect to see more G3 as well as G2 tumours. It is possible that upregulation of TNFα polymorphisms are associated with increased risk of the disease, but no overall increase in survival (Finally, there was no association of the TNF+488A or TNF−859T polymorphisms with recurrence or progression. This type of result has been found in other cancer studies of TNF polymorphisms. For example, in non-Hodgkin's lymphoma, although TNF polymorphisms have been shown to influence treatment outcome, progression-free survival and overall survival of patients (The natural history of G2 tumours is very heterogeneous and it is this group in particular that needs better prognostic information. A larger study would be needed to demonstrate whether TNF genetic variants influence prognosis within the G2 subgroup.α protein expression. If it affects the responsiveness of TNFα production to toxic stress, then this may explain the relation to risk of bladder cancer and higher grade and determine the effectiveness of intravesical BCG. Furthermore, there are now specific inhibitors of TNFα such as etanercept (In conclusion, we have demonstrated for the first time a TNF polymorphism association with risk of bladder cancer and grade of tumour at presentation. This is a further urological tumour association involving the polymorphism at position TNF+488, which has already been shown to be associated with prostate cancer. We have also demonstrated an additional TNF haplotype, which we hypothesise is of functional significance and which may help clarify the role of SNPs in influencing TNFanercept  and inflanercept  which coFurther investigation will require a molecular approach analysing the gene promoter in bladder epithelium.


Several studies have shown that cytotoxic T lymphocytes (CTLs) play an important role in controlling HIV/SIV infection. Notably, the observation of escape mutants suggests a selective pressure induced by the CTL response. However, it remains difficult to assess the definite role of the cellular immune response. We devise a computational model of HIV/SIV infection having a broad cellular immune response targeting different viral epitopes. The CTL clones are stimulated by viral antigen and interact with the virus population through cytotoxic killing of infected cells. Consequently, the virus population reacts through the acquisition of CTL escape mutations. Our model provides realistic virus dynamics and describes several experimental observations. We postulate that inter-clonal competition and immunodominance may be critical factors determining the sequential emergence of escapes. We show that even though the total killing induced by the CTL response can be high, escape rates against a single CTL clone are often slow and difficult to estimate from infrequent sequence measurements. Finally, our simulations show that a higher degree of immunodominance leads to more frequent escape with a reduced control of viral replication but a substantially impaired replicative capacity of the virus. This result suggests two strategies for vaccine design: Vaccines inducing a broad CTL response should decrease the viral load, whereas vaccines stimulating a narrow but dominant CTL response are likely to induce escape but may dramatically reduce the replicative capacity of the virus. As a result of their high mutation rate, HIV and its counterpart SIV in non-human primates can evade recognition by the host immune response through the generation of viral variants, the so-called escape mutants. This avoidance of cytotoxic T lymphocyte (CTL) mediated killing seems to be one of the major reasons why virus replication is not controlled effectively. However, it remains difficult to investigate the critical properties of the dynamics of immune escape. To this end, we developed a new computational model of HIV/SIV infection consisting of several CTL clones that can recognize specific parts of viral proteins, i.e., epitopes. The simulations allow us to follow the dynamics of immune escape in detail and help to interpret longitudinal data of HIV/SIV infections. Interestingly, changing the relative sizes of the CTL clones leads to a different evolution of the virus. Instead of reducing the number of infected cells, an alternative strategy of vaccine design could be to reduce the replicative capacity of the virus that might have implications for disease progression. HIV infection in humans and SIV infection in non-human primates is not cleared by the host's immune system. However, there is partial control of virus replication that is mainly attributed to the cytotoxic T lymphocyte (CTL) response It is a challenge to acquire longitudinal data on CTL escape since the virus diversity within a host has to be followed over a long phase of chronic infection. Additionally, little data is available during the early phase of infection, especially for HIV where the infection is difficult to diagnose during the first weeks. Asquith et al. The interaction of viral replication, mutation and selection by different CTL responses appears to be complex. Studies have observed CTL escape very early but also years after primary infection n different CTL clones recognizing n different epitopes derived from viral proteins. On the viral genome we allow two mutations to occur per epitope. One mutation confers escape from recognition by the specific CTL clone. Since escape mutations may be associated with a fitness cost in viral replication or infectivity, a second mutation can at least partially compensate for the fitness loss.We develop a computational model of HIV/SIV virus dynamics including a cellular immune response consisting of several CTL clones . We haveWe translate those interactions into a set of ordinary differential equations (ODEs) and add stochastic events for viral mutation. Classically, the processes of infecting target cells or the killing of infected cells by CTLs have been described with simple mass-action terms + target cells T are produced at a rate of λ cells per day, die at a rate δT and can become infected by virus particles of type iV with fitness if at a maximal rate of β per day. Target cell availability for virus particles is density dependent, as the infection rate per virus particle is saturating over the total number of CD4+ cells (non-infected and infected). After infection of a target cell, reverse transcription occurs during which the virus can mutate with a probability of μ per position  or 0 (no recognition). Following Michaelis-Menten kinetics, the CTL clones compete with each other for clearance of infected cells  they proliferate at a maximal rate g per day and die at a rate δE. Upon infection, virus replicates rapidly and typically iE we draw Non-infected CD4ed cells . When , The most surprising phenomenon of CTL escape in HIV/SIV is the time scale at which it occurs. Selection of escape variants has been found to happen very early after acute infection, but also late after years kh = 1012). kh = 109). In both simulations, the total number of infected cells peaks a few weeks after infection and reaches a set-point level of around 107 to 108 cells (black line in top panels) Our model describes the virus dynamics of an HIV/SIV infection and the subsequent immune escape in a very realistic manner . Many esiE are depicted in the bottom panels. Upon infection, the clones become stimulated depending on the parameter The dynamics of the CTL clones + target cell depletion It has been suggested that many escapes occur early, i.e. during the decline phase in viral load after the peak of infection and that they potentially prevent clearance of HIV/SIV. However, in our simulations immune escape does not occur before the set-point level is reached around two to three months after infection . This is−1Infected-cell death rates from different patients have been found to be close to a normal distribution with a mean of 0.45 dEscape variants not only appear at different times during infection but also with different rates. The rate at which an escape variant replaces the wild-type, the so-called escape rate, is determined by the balance between the evaded rate of killing and the fitness cost of the escape mutation −1, see −1, and most escape rates are below 0.2 d−1. The lower rates of escape are due to the fitness cost of escape mutations that cannot totally be restored by compensatory mutations. Interestingly, the rates of escape are not distributed equally over the time of infection  but rarely for low values of kh (saturation) (black dots).In etics hk . Althougetics hk , we can in vivo data one has to obtain ratios of escape variants to the wild-type. However, a problem arises when escapes are too rapid to be followed by the relatively long sampling intervals To estimate rates of escape from Our analysis shows several properties of the process of escape. Even though the total killing induced by all CTL clones is high, escape rates are expected to be slow. First, escape rates slow down due to the acquired fitness cost in viral replication, especially during later phases of infection. Secondly, if killing of infected cells follows Michaelis-Menten kinetics, the rates of escape are decreased. In addition, estimating rates of escape from ratios of escape variants and wild-type virus is likely to lead to an underestimation of the true rate. These findings highlight that slow escape rates do not necessarily infer low killing rates.−1) and is independent of CTL responses during HIV infection generally consist of several CTL clones recognizing many different epitopes. The size of those CTL clones can differ substantially resulting in dominant and sub-dominant responses  in size . Note, tTo investigate the effect of immunodominance we run simulations for different values of t escape . Concurrt escape . Howevert escape .The interaction of the HIV quasispecies with the CTL response appears to be complex which makes the analysis of experimental data difficult. Mathematical and computational models have been helpful to investigate how different processes influence each other. For example, a previous model described how shifting immunodominance and antigenic oscillations can occur during HIV infection Another explanation for the late appearance of escape variants that has been put forward is the waiting time for compensatory mutations Our simulations proved to be useful to analyze the process of escape. We conclude that, although killing can be very efficient, escape rates are expected to be low. The association with a fitness cost and the way how CTL clones interact with infected cells critically influence the rates of escape. Hence, it appears to be important to study those interactions to derive realistic killing terms Immunodominance affects the viral evolution within a host. A higher degree of immunodominance leads to more frequent escape with a reduced control of viral replication but a substantially impaired replicative capacity of the virus. This is interesting as vaccines generally aim to induce a broad CTL response where escape is unlikely to occur. However, a dramatic reduction of the replicative capacity of the virus due to escape could indeed slow down disease progression and/or reduce transmission The balance between the CTL response and the viral population acquiring escape mutations appears to be a dynamical process over a long period of infection. We have shown that it is important to analyze the kinetics of this process and also the time scale (acute and chronic phase) at which it occurs. More longitudinal data of HIV infections will help to further investigate this process and future research is likely to go into this direction.m(0), is 1. In the following, the ODEs are integrated using the routine odeint from Numerical Recipes t = 1 day, we approximate the number of cells that have been infected with virus of type i during the last time step according toμ. For the cells being newly infected with virus of type i, we calculate the integer number ijx of cells where the virus mutated into another type j. This is done by drawing from the binomial distribution ijx = B for each of the two positions (escape and compensatory) over a total of n epitopes. Then, the infected cell populations are updated accordingly (i.e. iI  = iI−ijx and jI  = jI+ijx). Whenever a previously not existing viral variant j is generated, m(t) = m(t−Δt)+1, and the set of ODEs is expanded. We also take into account the actual population size of virus infected cells The set of ordinary differential equations (ODEs) is extended with stochastic events for viral mutation (similar as in wtf = 1.0) and the higher fitness of the two single variants.Escape mutations in HIV and SIV are likely to confer a fitness cost in viral replication or infectivity w is the wild-type and e is the escape variant. Cells infected with wild-type virus replicate at a net rate a and are killed by all CTL clones recognizing epitopes other than the escape epitope at rate b. The CTL clone recognizing the escape epitope kills wild-type cells at rate c. Cells infected with the escape variant replicate at a net rate a′ and are only killed by the CTL clones recognizing epitopes other than the escape epitope. For c>0, the escape variant gains a growth advantage as long as the fitness cost of the escape, d = a−a′, is not higher (i.e. d<c). As shown in Asquith et al. http://sourceforge.net/projects/lmfit) based on the Levenberg-Marquardt algorithm to solve nonlinear least-squares problems. However, the fitting is likely to underestimate the true rates of escape as it is illustrated in The outgrowth of an escape variant and the subsequent replacement of the wild-type can be considered as a competitive growth between two populations:kh in Equation 8 we approach mass-action kinetics and the results are discussed.E is similar to the double-saturating term used in Equation 8: once the set-point is reached, −1 during the chronic phase of infection. I* and E* are the numbers of infected cells and CTL effector cells, respectively, in the steady-state in absence of escape. During the acute phase, we observe three types of dynamics: 1) For kh<5×107, the infection is always cleared since an infected cell can be killed at a rate of 7<kh<7×107, a very rapid escape during acute infection can prevent clearance of the infection. 3) For kh>7×107, we approach mass-action kinetics since kh>>I and the term becomes We assume Michaelis-Menten kinetics for the killing of infected cells by CTL effector cells:


This manuscript reports the production and preclinical studies to examine the tolerance and efficacy of an autologous cellular gel-matrix integrated implant system (IIS) aimed to treat full-thickness skin lesions.in situ gelification of fibrin into a porous crosslinked scaffold composed of chitosan, gelatin and hyaluronic acid. The in vitro cell proliferation within the IIS was examined by the MTT assay and PCNA expression. An experimental rabbit model consisting of six circular lesions was utilized to test each of the components of the IIS. Then, the IIS was utilized in an animal model to cover a 35% body surface full thickness lesion.The best concentration of fibrinogen and thrombin was experimentally determined by employing 28 formula ratios of thrombin and fibrinogen and checking clot formation and apparent stability. IIS was formed by integrating skin cells by means of the The preclinical assays in rabbits demonstrated that the IIS was well tolerated and also that IIS-treated rabbit with lesions of 35% of their body surface, exhibited a better survival rate .IIS should be further studied as a new wound dressing which shows promising properties, being the most remarkable its good biological tolerance and cell growth promotion properties. Natural polymers such as collagens, glycosaminoglycans, starch, chitin and chitosan have been used as biomaterials for skin substitutes because they closely resemble the native cellular milieu -3.An interesting new matrix was proposed by Liu et al, 2004[in vitro cultivation of artificial organs.Chitosan, a polysaccharide composed of glucosamine and N-acetyl glucosamine, obtained from N- deacetylation of chitin, is an excellent biomaterial due to its low cost, scale availability, anti-microbial activity and biocompatibility . It has The aim of the present research was to investigate the performance of a novel artificial skin chitosan- based scaffold containing autologous skin cells that have been included in a fibrin gel integrated inside the matrix. The ultimate goal of this research was to explore the tolerance and the efficacy of an IIS on an animal-based model.Ortolagus cuniculus rabbits were anesthetized with ketamine/xylasine (5 mg and 2 mg/100 g of body weight) [2 biopsy was taken. All the animal experiments and procedures, including animal procurement, surgery, anesthesia, euthanasia, animal housing and surgery facilities were performed by veterinarians following the Facultad de Farmacia-Universidad de Valparaíso animal care guidelines, which are based on the Guide for the Care and Use of Laboratory Animals from the National Research Council[ weight) . A selech Council.The technique used for isolation and culture of skin cells was adapted from previously published reports -19. BrieThe MTT  assay, which has been validated as a proliferation assay even inside microcarriers -23, was in situ gelification of the fibrin (100 μL of both thrombin and fibrinogen per square centimeter).The procedure described by Liu et al , was folThe best concentration of fibrinogen and thrombin was experimentally determined by employing 28 formula ratios of thrombin and fibrinogen.2) was selected to suspend a mixture of keratinocytes and fibroblasts. Fibroblasts and keratinocytes growing on separated T25 cell culture flasks (at passage 2 of primary cell culture) were tripsinized to recover both cell populations and seeded on the matrix to reach a final concentration of 3 × 104 cell/cm2 [The optimal formula ratio . Both systems had an average thickness of 3 mm and their shape and surface were tailored to the form of the skin lesion.Six circular 2,5 cm diameter full-thickness excision wounds were performed at the paravertebral skin of eight young-adult rabbits. For each of the lesions, the following treatments were applied: IIS, CFIS (cell free integrated system), fibrin, autologous skin cells in fibrin, porous matrix or no treatment. The position of the treatment on the dorsal area of the rabbit was randomly assigned.The performance of the treatments was evaluated by two blind referees, a medical doctor and a veterinarian. The outcome of each treatment was determined as graft take percentage, which is a clinical estimation of the area of the wound that is healed. Infection was categorized as a yes or no condition, and scar quality was scored based on color 1-5 scale), thickness  and wound retraction . The full description of the scale is summarized in Table -5 scale,In order to evaluate the efficacy of an IIS, a 35% full thickness body surface lesion was performed on young adult rabbits. Twelve duplets of rabbits from the same progeny paired by body weight were either treated with an IIS or left with no treatment. The rabbits were assigned at random to each condition. The outcome of the treatment was determined by survival, weight gain and wound closure efficiency.At the time of any wounds being clinically healed, treated or control, a biopsy of complete skin exceeding the initial size of the implant and including the region of wound healing, was taken. After fixing the biopsies in Bouin's solution for 24 h, they were rinsed in 70% ethanol, dehydrated until 95% ethanol, cleared in butanol, and embedded in Paraplast Plus . Serial sections 5 μm thick were cut in a Leica RM 2155 microtome. A selected set of sections was mounted on albumin-coated microscope slides and stained with a trichromic stain . The sections were stained in Harris Hematoxylin for only 75 seconds, rinsed in running tap water for 10 min, and then rinsed in distilled water. Next, they were stained in 0.5% erythrosine B (C.I. 45430) - 0.5% orange G (C.I. 16230) for 30 min, and then rinsed in distilled water. They were then immersed for 10 min in 0.5% phosphotungstic acid and then rinsed in distilled water. Finally, they were stained in 1% methylene blue (C.I. 42780) for 75 seconds, and quickly dehydrated in 95% ethanol followed by 100% ethanol. After clearing in xylene, the slides were cover-slipped with Poly-Mount Xylene mounting medium . Afterwards, photomicrographs were taken in a Leitz-Leica DMRBE microscope equipped with a Nikon Coolpix 5000 digital camera.2O2 in absolute methanol (to block endogenous peroxidase activity). After rinsing in 50 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (tris), pH 7.6 buffer, the slides were incubated with 2% normal horse serum in the same buffer and later incubated overnight at 4°C in the monoclonal antibody to Proliferating Cell Nuclear Antigen  ; the sections were subsequently incubated with biotinylated antimouse IgG (1/500) and then processed using peroxidase-ABC  amplification procedure and DAB  as chromogen, and finally were slightly counterstained with Harris Hematoxylin for 10 seconds.For the PCNA immunochemical analysis, deparaffinized and rehydrated integrated implant system (IIS) sections were incubated for 5 min in a microwave oven ; then cooled down to room temperature, rinsed in distilled water and incubated in 3% HTwo variables were compared after 10 days post implant for the IIS and CFIS: percentage of graft take and scar color using the 1-5 ranking described in the above methods. Both variables are not normally distributed, therefore a standard nonparametric method was applied as described by Hollander and Wolfe .Pairs of rabbits were assigned at random to treatment with an IIS or to a control group. Success, which was assessed as the rabbit surviving, was compared between both groups, applying the Mc Nemar Test ,26. The Twenty-eight different formulations changing fibrinogen and thrombin concentration ratio were evaluated for clotting formation and apparent stability as shown in Figure From the immobilization zone, five formulae were selected comprising a wide range of fibrinogen and thrombin concentration ratio. These five formulae were evaluated for stability, by incubating the clot in fresh cell culture media and in conditioned media, recovered from preconfluent skin cell cultures.The optimal formula was chosen to be 13 mg/mL fibrinogen and 130 NIH/mL thrombin, which yielded fair clots that underwent fibrinolysis close after 24 h Figure , since iIn Figure The cell proliferation within the IIS was examined by the MTT assay and compared with cells grown in a conventional culture flask Figure . After 7Clinical evaluation in rabbits showed that after 10 days post implant, the graft take of CFIS was 53.75% and 81.25% for the IIS, demonstrating a significant statistical difference (p < 0.10). Evidence of mild infection was reported in 2 out of 8 rabbits for the CFIS, and also 2 out of 8 rabbits for the IIS. Scar color after 10 days was rated as 1.9, for the CFIS and 2.75 for the IIS, demonstrating a significant statistical difference (p < 0.05). There were no significant differences in the wound thickness or retraction of the scar during the evaluation period between the CFIS and IIS treated rabbits.After 60 days, the wound surface was completely closed according to clinical evaluation, indicating that full epithelization occurred. A biopsy of the treated area was taken from each animal and processed as described in the above methods. Typical histological results are shown in Figure It is important to highlight that at the time of the biopsy there were no signs of blood inflammatory cells like neutrophils, lymphocytes or macrophages, neither abscess or discharge of some kind of exudates such as serous, seropurulent, haemopurulent or pus, in any of the treated rabbits, typically associated with infection or rejection.Rabbits which survived a 35% body surface lesion after an IIS treatment were compared with the untreated group utilizing the MacNemar test. This analysis indicated that the rabbits subjected to an IIS exhibited a better survival rate compared to the control group (p = 0.06).The area of cicatrisation, of eleven pairs of rabbits was compared. One couple had to be withdrawn from this analysis because they died before five days of intervention.The use of the Shapiro-Wilk procedure showed that the difference of area of cicatrisation is not rejected for the normality assumption required to use the paired t test (p > 0.55). This test indicated that the area of cicatrisation (open wound) of the IIS treated rabbits was significantly (p = 0.06) smaller than the control rabbits. In addition, a sub analysis was performed using just the pairs of rabbits in which neither the control nor the case rabbit died. In four pairs of rabbits, the control rabbits died before the end of the study . With the seven pairs of rabbits that survived at least 50 days, the difference in the area of cicatrisation among ISS treated rabbits and control rabbits exhibited a higher statistical significance (p < 0.02).In addition, the majority of the rabbits in the IIS treated group showed a better curve of weight gain than the non treated animals Figure . The difThis article describes a skin implant system built based on a novel approach that better integrates cells to a polymeric scaffold using fibrin as the cell carrier. The IIS was developed with the purpose of creating a wound dressing for regeneration of skin damaged by burns or other severe trauma. The IIS has the benefit of combining the presence of cells that has been reported by some authors as helping the healing process with fibrin which is a known natural component found in injured tissue at early stages of wound repair ,27 and ain vitro [It is well known that the interaction between cells and the physical surface of cell culture play an important role in the outcome of the cells, influencing biological processes such as cell proliferation and differentiation. The developed system allows the cells to proliferate noticeably better than cells growing in conventional cell culture flasks. This might explain the positive clinical outcome of the IIS, since in a very short time after seeding, it improves cell proliferation within the system, a critical condition required to successfully treat severely injured skin. The observed high increase in cell proliferation may be the result of the cells actively secreting growth factors, such as PDGF , which min vitro . The ligin vitro . PDGF pain vitro . In addiin vitro . Thus, tIn agreement with other authors, the results presented, also point towards the beneficial effects of the presence of cells in the scaffold used for dermal restoration in the wound healing process ,42. BeneThe use of fibrin in dermal substitutes has shown several benefits like adjuvant of hemostasis and graft take. In addition, there is experimental evidence of fibrin acting as an antibacterial agent, a major challenge to the success of the dermal substitute. The mechanism of antibacterial action has been partially attributed to the stimulation of phagocytosis . DespiteThe preclinical experimental evidence supports that the IIS is well tolerated and efficacious because there were no signs of inflammation and all the wounds healed, showing complete epithelization. Moreover, when a life threatening lesion was performed, the IIS treated animals exhibited an overall better survival, better growth over time and smaller cicatrisation areas.The use of autologous cells in this system is an advantage, not only because a scar of better quality is achieved, but also because it minimizes the infectious diseases transmission risk from one individual to another. However, the use of autologous cells might be seen as a drawback in view of the difficulties to store and transport of living cells and also higher costs due to their reduced possibilities of scale economy. Nonetheless, there is an autologous skin substitute currently available in the market, as well as, autologous dermal treatments for other applications. The use of animal-derived components, such as gelatine and fibrinogen, could be seen as a potential risk of transmission of certain animal borne infections, however, these and all of the components of IIS are available as pharmaceutical or tissue grade materials, presenting a risk which is comparable with products already within the pharmaceutical market.The preclinical assays reported here show especially encouraging findings to continue with standardized clinical trials for the IIS and also to continue investigating the cell-biomaterial-skin interaction.in vitro cell growth and constitutes a medical device that promotes wound healing at the preclinical level.An IIS is a wound dressing composed of known biomaterials combined in a novel approach, allowing the integration of the cellular component within the porous matrix. This gives the cells a microenvironment which promotes CFIS: cell free implant system; DMEM: Dulbecco's Modified Eagle's Medium; DMEM/F12: Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F12; EDC: 1-ethyl- carbodiimide; EDTA: ethylenediaminetetraacetic acid; IIS: cellular gel-matrix integrated implant system; MES: 2-morpholine-ethane sulfonic acid; MTT: 3--2,5-diphenyltetrazolium bromide; NHS: N-hydroxysuccinimide; PBS: pH 7.4 0.1 M phosphate buffered saline; SDS: sodium dodecyl sulfate.Young ME, Weinstein-Oppenheimer CR, Aceituno A, Acevedo C, Brown D and Tapia SM have a patent application for IIS.CWO: designed the scaffold, IIS and protocols for cell culture. She worked on the draft of the manuscript. ARA: designed and prepared the scaffold. He worked on the draft of the manuscript. DIB: performed the histological analysis and preclinical assay design and analysis. He worked on the draft of the manuscript. CA: designed cell viability assays and carried out fibrin/thrombin ratio experiments. He worked on the draft of the manuscript. RC: carried out the cell culture and cell viability assays. MAF: implemented the cell cultures and assembly of the IIS. FA: designed and prepared the IIS. CHR: performed statistical design and analysis of the preclinical assays. He worked on the draft of the manuscript. PM: was involved with the preclinical assays design, its performance and analysis. CM: participated in the design of the preclinical assay. SMT: participated in the design of the preclinical assay. MEY: developed the IIS and participated in the preclinical assay design. He worked on the draft of the manuscript.All authors read and approved the final manuscript