Elevated cyclic adenosine monophosphate (cAMP) enhances the barrier integrity of the corneal endothelium and thereby facilitates stromal hydration control, which is necessary for corneal transparency. This study investigates whether elevated cAMP is effective against the tumor necrosis factor-alpha (TNF-α)-induced loss of barrier integrity in monolayers of bovine corneal endothelial cells (BCEC).BCEC in primary culture were used for the study. Trans-endothelial electrical resistance (TER), a measure of barrier integrity, was determined by electrical cell-substrate impedance sensing. The changes were also ascertained by measuring paracellular permeability to fluorescein isothiocyanate (FITC)-dextran (10 kDa) across cells grown on porous culture inserts, and by immunofluorescence imaging of the apical junctional complex (AJC). The activation of p38 MAP kinase was assessed using western blotting.Co-treatment with forskolin, which activates adenylate cyclase, and rolipram, which inhibits cAMP-dependent phosphodiesterase PDE4, reduced the TNF-α-induced increase in the flux of FITC-dextran. Similar co-treatment also prevented the TNF-α-induced disorganization of zona occludens-1 (ZO-1) and cadherins at the AJC. Co-treatment, as well pre-treatment, with forskolin plus rolipram prevented the TNF-α-induced decrease in TER. The influence of the agents was significant after 12 h of exposure to the cytokine. This effect was also mimicked by A2B agonists, adenosine and 5′-N-ethylcarboxamidoadenosine (NECA), which are known to mobilize cAMP in BCEC. Elevated cAMP also inhibited the cytokine-induced activation of p38 MAP kinase, and further blocked the disassembly of microtubules as well as the disruption of the PAMR  at the AJC.These results suggest that elevated cAMP opposes the TNF-α-induced loss in barrier integrity of the corneal endothelium. This effect follows inhibition of the cytokine-induced activation of p38 MAP kinase and its downstream signaling involved in the disruption of AJC and PAMR, as well as the disassembly of microtubules. Corneal transparency depends on stromal deturgescence, which is maintained by the ‘fluid pump’ activity of its posterior monolayer, the endothelium -4. A majAdenosine and forskolin, agents known to elevate cAMP in the corneal endothelium, are known to promote stromal deturgescence in rabbits . Specifi7 U/mg; endotoxin free), forskolin, adenosine, 5′-N-ethylcarboxamidoadenosine (NECA), rolipram, pan-cadherin antibody, α-tubulin antibody, and FITC-dextran were purchased from Sigma Aldrich . Texas Red-conjugated phalloidin, goat anti-mouse Alexa-488, and anti-fade agent were purchased from Molecular Probes . The ZO-1 antibody was procured from Zymed . Electrodes (8W10E+) for TER measurements were purchased from Applied Biophysics, Inc. . Tissue culture inserts (0.2 μm pore size) were obtained from Nunc . The enhanced chemiluminescence kit for western blotting analysis was obtained from Thermo Scientific .TNF-α .Primary cultures of BCEC from fresh eyes were established as described previously -19. The The components of the cytoskeleton and apical junctional complex (AJC) were stained using an immunocytochemistry protocol, as described previously -31. Cell5 cells/ml on gold electrodes and grown to confluence. After the monolayers showed steady resistance values, they were exposed to desired agents after at least 1 h exposure to serum-free medium. The resistive portion of the measured impedance (TER), normalized to its initial value, was monitored continuously and taken as a measure of barrier integrity.The TER was measured using the principle of electric cell-substrate impedance sensing using a commercial device , as described previously ,27-31. C™; Fisher Scientific, Pittsburgh, PA) to confluence. The monolayers were then serum-starved for 1 h and either left untreated or exposed in triplicate to desired agents. Following treatment, FITC-dextran (10 kDa), dissolved in Ringers, was introduced in the apical compartment at a concentration of 0.4 μg/ml. After incubation at 37 °C for 2 h, samples were taken from the basolateral chamber for fluorescence measurements. Fluorescence was measured using excitation at 492 nm and the emission was collected at 520 nm.The changes in permeability to FITC-dextran were determined by a protocol, as described previously -31. CellConfluent monolayers were serum-starved overnight before treatment with desired agents. The cells were then lysed with NP-40 lysis buffer  containing protease inhibitor cocktail  and 5× reducing sample buffer . The lysates were transferred to a microcentrifuge tube kept on ice, followed by sonication for 10–15 s. The lysates were centrifuged at 14,000× g for 10 min followed by heating at 95–100 °C for 5 min. The samples were cooled to RT, centrifuged, and resolved by electrophoresis. This was followed by transfer onto a nitrocellulose membrane and incubation with the primary antibody  overnight at 4 °C. The blots were washed with wash buffer, followed by incubation with HRP-conjugated secondary antibody for 1 h at RT. The blots were visualized using an enhanced chemiluminescence kit . After visualization, the blots were stripped of phospho-p38 antibody and probed for total p38 MAP kinase with the anti-p38 MAP kinase antibody .Statistical comparisons were made using one-way ANOVA (ANOVA) with Bonferroni’s post-test analysis using GraphPad Prism software . A value of p<0.05 was considered statistically significant. Data are expressed as mean ±SEM. The “n” represents the number of independent experiments.We first examined the influence of cAMP on (TNF-α)-induced increase in the paracellular permeability to FITC-dextran. Exposure to 20 ng/ml of TNF-α for 20 h in the apical chamber increased the flux of FITC-dextran by >8 fold when compared to untreated cells . HoweverThe influence of TNF-α on the integrity of tight junctions (TJs) and adherence junctions (AJs) was examined by immunostaining ZO-1 and cadherins as markers, respectively ,33. In uTo assess the influence of elevated cAMP on TNF-α response at a higher temporal resolution, we reexamined the barrier integrity in terms of TER, as measured using the principle of electrical cell-substrate impedance sensing . As demoRecently, we showed that p38 MAP kinase is activated in response to TNF-α . The actIn our previous study , we obseCorneal endothelial dysfunction due to aging, iatrogenic injury, Fuch’s dystrophy, and inflammation is known to induce stromal edema leading to loss of acute vision ,8. CurreIn our first study on TNF-α , we demoIn our first series of experiments, the barrier integrity was assessed by following the flux of FITC-dextran. A nearly eightfold increase in permeability to the dextran was reduced to only twofold when cells were co-treated with forskolin and rolipram . Since . SpecifiAs shown in In our previous studies ,29, we oAlthough (TNF-α)-induced barrier dysfunction has been widely examined ,37,38, tThe other major mechanisms implicated in the response to TNF-α include activation of ROS/RNS (reactive oxygen/nitrogen species) -47, streIn conclusion, we have demonstrated for the first time that elevated cAMP opposes p38 MAP kinase activation by TNF-α, and thereby attenuates downstream events leading to barrier dysfunction in the corneal endothelium. Hence, elevation of cAMP can be a therapeutic strategy to overcome endothelial barrier dysfunction in response to TNF-α during corneal transplantation failure and/or uveitis.


TNF-α)−308 G/A polymorphism and pseudoexfoliation (PEX) glaucoma.To investigate the possible association between tumor necrosis factor alpha . The A polymorphic allele frequency was 3.2% in patients with PEX compared with 8.2% in controls (p=0.023).TNF-α−308 G/A genotype is not associated with PEX glaucoma. The −308G/A variant may be a possible protective factor against PEX.Our results suggest that  TNF-α gene polymorphisms at position −308 and −238 are the best characterized polymorphisms and have been demonstrated to influence TNF-α expression [TNF-α  also results in higher TNF-α production [TNF-α−308 gene polymorphism in patients with PEX glaucoma.Glaucoma is characterized by a progressive loss of retinal ganglion cells. A previous study suggests that tumor necrosis factor alpha (TNF-α) may contribute to the disease process . Pseudoepression .The G tooduction . In thisThis case-control study included 110 patients with unilateral or bilateral PEX glaucoma, and 110 control subjects. The control group and PEX patients were selected from the General Ophthalmology Polyclinic and Glaucoma section, respectively . On ophthalmologic examination, the anterior and posterior segments of all the cases were evaluated, and intraocular pressures (IOP) were measured with Goldmann applanation tonometry. The corneal endothelium, iris, and iris margins were evaluated for pseudoexfoliative material before dilation, and after dilation, the anterior lens surface was examined. To evaluate the angle for pseudoexfoliative material and increased pigmentation, we used gonioprisms. Criteria for inclusion in normal subjects were IOP less than 22 mmHg, normal visual fields, no evidence of glaucomatous changes in optic disc, and no history of glaucoma or ocular hypertension in first-degree relatives. These controls had no pseudoexfoliative material at the anterior lens capsule or pupillary margin. Unilateral or bilateral PEX glaucoma was diagnosed by the presence of PEX in the anterior segment, elevated intraocular pressure (>21mmHg), typical glaucomatous optic nerve changes , and glaucomatous visual field defects. All of the patients and control subjects were Turkish. Patients and controls were informed of the experiment including a detailed explanation of all the procedures and gave their consent. The study was conducted in accordance with the principles of the Declaration of Helsinki.2, and 25 pM of each of the primers. Cycling conditions were as follows: initial denaturation at 94 °C for 5 min followed by 35 cycles at 94 °C denaturation for 30 s, 55 °C annealing for 30 s, and 72 °C extension for 30 s. A final extension was performed at 72 °C for 7 min. The product was electrophoresed in a 1% (wt/vol) agarose gel, and bands were visualized under ultraviolet (UV) after ethidium bromide staining.Venous blood samples were taken from all participants. Genomic DNA was isolated from peripheral blood by conventional phenol-chloroform method. The polymerase chain reaction (PCR) method was described in previous studies ,9. A 194To determine the A allele at position −308, 12.5 μl of the PCR product was digested overnight with 10 U of the enzyme, BspHI, at 37 °C and electrophoresed in a 2.5% (wt/vol) agarose gel. This digestion results in an uncut 194 bp fragment when G is present at −308 position and in two fragments of 169 bp and 25 bp when A (TNF2) is present.2 test, and the odds ratio was calculated. Age was compared between groups by Student’s t-test. Allele frequencies were determined by gene counting. To assess the agreement between genotypes observed and those predicted by the Hardy–Weinberg equilibrium, the likelihood ratio test (G statistic) was used [2 test with a significance level (α) of 0.05. A p value less than 0.05 was considered statistically significant.Nominal variables were evaluated by the χwas used ,12. A saTNF-α G/A polymorphism was in accordance with the Hardy–Weinberg equilibrium in the PEX patients with glaucoma and the controls . The distribution of TNF-α G/A polymorphism between PEX glaucoma patients and controls was significantly different from each other (p=0.019). The frequency of the GG genotype in the PEX glaucoma group was higher than that of controls  in Chinese patients. They determined that the distribution of TNF-α−308 G/A was significantly higher in the POAG patients. Mossbock et al. [TNF-α−308G/A polymorphisms did not significantly differ between Caucasian patients with POAG and control subjects. Funayama et al [TNF-α-308 G/A genotype between Japanese patients with glaucoma  and healthy subjects.Lin et al.  evaluatek et al.  concludema et al . did notTNF-α−308 polymorphism in Turkish PEX glaucoma patients. TNF G/A genotype was also found to be lower in our PEX glaucoma patients than in control subjects, we detected a high prevalence of the G/G genotype in Turkish PEX glaucoma patients. However, our main hypothesis was to demonstrate the higher frequency of the GA genotype in PEX glaucoma patients. To our knowledge, this is the first study that evaluated the association between the TNF-α polymorphism and PEX glaucoma. It is interesting that the TNF-α−308 A allele frequency is similar to previous studies from Turkey in different age groups [TNF-α-308 G/A polymorphism has been studied in association with different complex diseases. Although increased TNF-α expression is linked to several diseases, some studies similar to ours have reported opposite results. The exact etiology of psoriasis is still unclear but the increased levels and activity of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α were suggested in the development of psoriasis [In this study, we investigated the distribution of the e groups ,24. Furtsoriasis . Howeversoriasis  performesoriasis . Parra-Rsoriasis  detectedsoriasis  performeTNF-α−308 G/G may be a genetic marker for identifying patients with increased risk of PEX glaucoma. Exactly how the GG polymorphism works to influence risk for PEX glaucoma remains unknown. Probably the genetic background of our population may be the reason for higher frequency of GG genotype in PEX glaucoma. Further studies are needed to confirm this finding.In our study, the GG genotype was higher in PEX glaucoma patients than in healthy controls. In conclusion, we could not find significant association between the 308 G/A genotype and the susceptibility to PEX glaucoma.


The thymus was normal inTNF/LTα-/- mice with regard to cell yields and stromal architecture. Detailed analysis of αβand γδ T cell-lineage thymocyte subsets revealed no abnormalities, implying that neither TNFnor LT play an essential role in T cell differentiation or positive selection. The number anddistribution of thymic CD11c+ dendritic cells was also normal in the absence of both TNF andLTα. A three-fold increase in B cell numbers was observed consistently in the TNF/LTα-/-thymus. This phenotype was due entirely to the LTα deficiency and associated with changesin the hemopoietic compartment, rather than the thymic stromal compartment of LTα-/- mice.Finally, specific Vβ8+ T cell deletion within the thymus following intrathymic injection ofstaphylococcal enterotoxin B (SEB) was TNF/LT independent. Thus, despite the presence ofthese cytokines and their receptors in the normal thymus, there appears no essential role foreither TNF or LT in development of organ structure or for those processes associated with Tcell repertoire selection.TNF, lymphotoxin (LT) and their receptors are expressed constitutively in the thymus. Itremains unclear whether these cytokines play a role in normal thymic structure or function.We have investigated thymocyte differentiation, selection and thymic organogenesis in genetargeted mice lacking LTα, TNF, or both (TNF/LTα


Tumour-necrosis factor (TNF) is an anti-tumour factor released into the serum of BCG-primed rabbits after i.v. injection of endotoxin. Although negligible amounts of TNF are produced in normal, unprimed animals after endotoxin injection, monocytes from these rabbits can produce TNF after endotoxin challenge in vitro. This paper (a) establishes the optimal conditions for TNF production in vitro by mononuclear phagocytes from various tissues and (b) compares tissues from normal and BCG-injected rabbits for TNF production in vitro. Optimal amounts of TNF are produced by mononuclear phagocytes in the presence of endotoxin. The TNF is newly synthesized, mainly in the first 7 h of culture, and has similar gel-filtration and ion exchange behaviour irrespective of its source. For both normal and BCG-injected rabbits, alveolar and peritoneal macrophages are the most potent producers, followed by blood monocytes, spleen macrophages and marrow cells. The liver is also an important site of TNF synthesis. In the tissues of BCG-injected rabbits there are more mononuclear phagocytes than in normal rabbits, and these cells have enhanced capacity to produce TNF. Taking both factors into account it can be calculated that, after injection of endotoxin in vivo, over 20 X more TNF would be produced by BCG rabbits than normal rabbits, assuming that the major sources of production are the lungs, blood, spleen and liver.


N-ethyl-N-nitrosourea) mutagenesis is a useful way of generating models. We have characterised a new ENU-induced mouse mutant, Oblivion , showing semi-dominant inheritance of hearing impairment. Obl/+ mutants showed increasing hearing impairment from post-natal day (P)20 to P90, and loss of auditory function was followed by a corresponding base to apex progression of hair cell degeneration. Obl/Obl mutants were small, showed severe vestibular dysfunction by 2 weeks of age, and were completely deaf from birth; sensory hair cells were completely degenerate in the basal turn of the cochlea, although hair cells appeared normal in the apex. We mapped the mutation to Chromosome 6. Mutation analysis of Atp2b2 showed a missense mutation (2630C→T) in exon 15, causing a serine to phenylalanine substitution (S877F) in transmembrane domain 6 of the PMCA2 pump, the resident Ca2+ pump of hair cell stereocilia. Transmembrane domain mutations in these pumps generally are believed to be incompatible with normal targeting of the protein to the plasma membrane. However, analyses of hair cells in cultured utricular maculae of Obl/Obl mice and of the mutant Obl pump in model cells showed that the protein was correctly targeted to the plasma membrane. Biochemical and biophysical characterisation showed that the pump had lost a significant portion of its non-stimulated Ca2+ exporting ability. These findings can explain the progressive loss of auditory function, and indicate the limits in our ability to predict mechanism from sequence alone.Progressive hearing loss is common in the human population, but we have few clues to the molecular basis. Mouse mutants with progressive hearing loss offer valuable insights, and ENU ( Atp2b2, which encodes a calcium pump. We have discovered a new mutation in this gene leading to hearing loss in the mouse mutant oblivion. The mutation leads to a serine to phenylalanine substitution in a transmembrane domain. Mutations affecting such transmembrane domains are usually expected to interfere with the normal process of inserting the protein in the membrane and transporting it to its final destination on the plasma membrane of the cell. Surprisingly, the pump is produced and is targeted to the plasma membrane, in both cultured cells expressing the mutant gene and sensory hair cells from the oblivion mutant inner ear. However, we show it has impaired calcium pumping ability, which can account for the progressive hearing loss as well as the progressive degeneration of the sensory hair cells that we observe in the mutants.Progressive hearing loss is very common in the human population, but we know little about the causes. Environmental and genetic factors each may contribute. Knowledge of the genetic variants involved in hearing loss and understanding of the molecular and cellular mechanism of their action will aid the development of better treatments. One of the few genes known to be involved, in both mouse and humans, is  The C-site insert leads to a truncated pump that contains only about half of the original calmodulin binding domain w/a variant, seen in stereocilia, has an unusually limited ability to increase activity rapidly when challenged with a Ca2+ pulse, but has about the same high non-stimulated activity as the full-length z/b variant Transcripts for PMCA2 undergo alternative splicing at two sites. Site A is closer to the N-terminus and site C closer to the C-terminus. In the PMCA2 variant expressed in stereocilia Obl) mutant was identified as a new mouse mutant with progressive hearing loss from a large scale ENU mutagenesis screen Obl is due to a missense mutation in the gene Atp2b2, encoding PMCA2. We describe the hearing impairment and hair cell pathology in the mutants, the dysfunction of Ca2+ export by the mutated PMCA2 pump cloned and overexpressed in model cells and in cultures of utricles from the mutant mice.The Oblivion , a reflection of cochlear and brainstem neural activity, were recorded in P20, P59–62 and P89–91 mice on their original C3HeB/FeJ genetic background . ABRs ofObl/+ mice demonstrated a severe and age-related progressive hearing loss. Obl/+ mice showed significantly raised thresholds at all frequencies, compared to age-matched wild-type controls , of up to 60–70 dB or more. In contrast to the Preyer reflex tests, even at P20 Obl/+ showed large threshold elevations. At P59–62, the heterozygotes showed further threshold elevations which were most severe at higher frequencies, above 18 kHz. By P89–91, high frequency losses were compounded by severe losses across the entire range measured. This indicated a progressive hearing loss in Obl/+ mice. The improvement of low frequency (3–6 kHz) ABR thresholds between P20 and P59–62 may indicate maturation of the developing auditory system between these ages.Obl/Obl homozygous mutants show a very severe hearing and vestibular phenotype and are significantly smaller  than age and sex matched Obl/+ littermates . They (a) fail to develop a Preyer reflex; (b) fall from side to side whilst walking; (c) are unable to right themselves; and (d) curl towards their belly when lifted by their tail and do not display a reaching response when lowered towards a surface. Homozygotes also show hind limb stiffness and appear ataxic, which are not general features of vestibular dysfunction.Obl/+ mutants at 3–4 months of age showed degeneration of hair cells, with the basal turn more severely affected than the apex, and outer hair cells (OHCs) more affected than inner hair cells (IHCs), a pattern that is commonly reported in damaged cochleas  in exon 15 of Atp2b2, predicted to change a serine to a phenylalanine   in control, 6.55±0.72 sec (n = 9) in the wild type w/a variant and 45.50±5.97 sec (n = 4), p<0.001, in Obl.To investigate the effects of the serine to phenylalanine change on the functionality of the pump, mammalian expression plasmids for the S877F and the wild type variant of the PMCA2 pump were prepared and expressed in CHO cells. Appropriate controls (Western blotting and quantitative immunocytochemistry) established that the two pump variants were expressed at about the same levels, and were correctly delivered to the plasma membrane . CHO cel cytAEQ,  and stimRCA) see  and the a2+ peak . The mut2+ dynamics in the stereocilia of hair cells, cultures of immature utricular maculae were obtained from wild type and mutant mice (see 2+-dependent changes in fluorescence evoked by the photorelease of intracellular caged Ca2+ (4 ns single UV pulse) were monitored with a temporal resolution of 6 ms using confocal laser scanning microscopy. Immunofluorescence labelling with isoform specific antibodies showed that PMCA2 was correctly located in the stereocilia of homozygous mutant organotypic cultures of utricular maculae (F), normalized to basal (pre-stimulus) fluorescence (F0), are compared for wild type  heterozygous Oblivion mice  and homozygous Oblivion mice  in 2+]i transient decayed more slowly for the Obl/Obl mice. To highlight the differences, the traces are re-plotted in Obl/+ and Obl/Obl cultures: τwt = 2.8±0.4 s for wt, τObl/+ = 4.2±1 s for Obl/+ mice  and τObl/Obl = 6.9±1.3 s for Obl/Obl mice (p<0.01). No significant divergence in the early phase (first 2 s) of the decay was found for Obl/+ mice relative to wt, consistent with the lack of evident phenotypic vestibular defects in these mice. At later times, the decay tended to diverge possibly due to the contribution of complex processes such as Ca2+-induced Ca2+ release. These contributions were not investigated further.To characterize Camice see . Ca2+-de maculae  and orga maculae . Figure Progressive, age-related hearing loss affects 60% of humans over the age of 70 Atp2b2, OblAtp2b2 in the Obl mouse mutant. This allele contains a C to T missense mutation in exon 15 of Atp2b2, causing a non-conservative amino acid substitution of serine by phenylalanine in transmembrane domain 6 of the PMCA2 pump Obl pump was correctly delivered to the plasma membrane in both the model cells and in native stereocilia. In another Atp2b2 mutant allele, Wriggle mouse Sagami (wri), a missense mutation in transmembrane domain 4 completely abolished the expression of PMCA2 protein in stereocilia of cochlear hair cells 2+ is translocated The present study has identified a new ENU-induced allele of Obl mutation is the seventh mutation in mouse Atp2b2 to be reported. In the deafwaddler (dfw) mouse a missense mutation in Atp2b2 leads to a partial loss of function 2+ pumping activity Atp2b2 led to clear ataxic behaviour, with normal mRNA levels, in a second unnamed mutant Atp2b2 have been described: deafwaddler 2J (2Jdfw), deafwaddler 3J (3Jdfw), wriggle mouse sagami (wri) and a targeted null mutation. Analysis of mRNA transcripts and protein levels suggests that these latter four mutations are all null alleles Obl homozygotes is similar to the phenotype of these other known Atp2b2 null mutants Obl and dfw confirmed that these two mouse mutants are likely to be allelic. The mutations identified in the Atp2b2 gene are shown in The Obl and the dfw mutations significantly reduce the non-activated Ca2+ exporting ability of the PMCA2 protein. Observations on organotypic cultures showed that the defect of the pump observed in model cells also reduced its ability to remove the Ca2+ produced by UV photolysis in stereocilia. Thus, the Obl mutant has lost a significant portion of the non stimulated, longer-term Ca2+ exporting ability in respect to the w/a PMCA2 pump.Both the Obl/+ mice is detected at P20 and progresses in its severity with increasing age. Threshold shifts of up to 60-70 dB were found at frequencies corresponding to the basal and middle turns of the cochlea, where the majority of outer hair cell loss was detected. Analysis of Obl/+ mice at P20 showed no OHC or IHC loss despite the fact that they had a significant hearing impairment, confirming the previous suggestion that although hair cells are present they are not functioning normally In the present work we show that hearing loss in Obl/+ mutants at 4 months of age. The early degeneration of the OHCs seen in heterozygous Obl (and dfw) mice, leading to complete degeneration of the organ of Corti in the base of the cochlea, is similar to that seen in some human patients with age-related hearing loss dfw mice at approximately P60, distortion product otoacoustic emission studies show that PMCA2 is important for the correct functioning of outer hair cells, especially at high frequencies dfw2JAtp2b2 allele, Obl/+ mice are elevated above controls, but are still recordable. An interesting observation from the ABRs recorded in P59–62 and P89–91 Obl/+ mice is that click thresholds are more sensitive than the best tone threshold, by a factor of 15–22 dB. Clicks produce a more synchronised activation of a wider region of the basilar membrane compared to tone pips, and this may produce a summation of activity, reflected in lower click-evoked ABR thresholds.IHC loss was detected in the base and middle of the cochlea in Obl was placed on a mixed C3HeJ/FeB and C57BL/6J background, the progressive hearing loss in heterozygotes seemed more severe (data not shown). Onset and severity of progressive hearing loss seen in heterozygotes of other Atp2b2 mutant alleles have been shown to vary considerably depending on the genetic background on which the mutation arose Cdh23 at the ahl locus  Atp2b2 mutations and Cdh23 mutations have been shown to worsen the progressive hearing loss seen in some human patients. Heterozygous mutations (V586MATP2B2) increase the severity of the progressive hearing loss seen in human patients with mutations in CDH23V586MATP2B2 mutation reduced the level of PMCA2 protein produced by 50%, although on its own it was not sufficient to cause hearing impairment in humans G293SATP2B2 mutation and a T1999SCDH23 mutation. The parents of the patient carried either the ATP2B2 or the CDH23 mutation, but had no hearing impairment When ATP2B2 gene has recently been implicated in the deafness in 3p- syndrome. The syndrome is characterised by developmental delay, growth retardation and craniofacial abnormalities  arose from the ENU mutagenesis program at Munich N-ethyl-N-nitrosourea (ENU) into C3HeB/FeJ males. F1 progeny were screened for a range of phenotypes, including deafness and balance disorders. The founder Obl/+ mutant was identified by the absence of a Preyer reflex at 3 months. The mutant colony was maintained on the C3HeB/FeJ background, and all ABR and structural analysis was carried out on this genetic background. The care and use of animals was carried out in accordance with UK Home Office regulations and the Animal Care and Use Committee of the University of Padua.The founder mouse carrying the Oblivion mutation  and middle turn (55–65% from the base) at 20 (P20), 72–75 and 121 days old. Between 3 and 7 Obl/+ and +/+ mice were examined at each age. Hair cells with intact stereocilia bundles were counted over a stretch of at least 200–300 µm of the cochlear duct. Hair bundles that were damaged or showed fusion were still counted as being present. A two tailed T-Test was performed on hair cell counts for each hair cell row in each region at 95% confidence intervals, with the number of cases and standard deviations included in the analysis. A two tailed T-test was carried out on the weights of Obl/Obl mutants compared to littermate (Obl/+) controls in the same way.Inner ears were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, the organ of Corti was exposed and samples were processed using the osmium tetroxide-thiocarbohydrazide (OTOTO) method Obl/+×+/+ matings were tested weekly from 3 to 8 weeks of age, although the numbers of mice at each time-point varied. For ABR recordings, a separate cohort of mice were anaesthetised (urethane 2 mg/g) and subcutaneous needle electrodes inserted on the vertex (active), and over the left (reference) and right (ground) bullae. A calibrated sound system was used to deliver free-field click (0.01 ms duration) and tone pip  stimuli at a range of intensity levels in 3 dB (or multiple) steps. Averaged responses to 512 stimuli, presented at 21.1 s−1, were analysed and thresholds established as the lowest sound intensity giving a visually-detectable ABR response. ABR recordings were obtained from a total of 61 mice, 28 at P20 , 14 at P59–62  and 19 at P89–91 .The Preyer reflex (pinna flick) was detected using a custom-built click box to deliver a calibrated 20 kHz sound burst at 90 dB SPL. Up to 88 mice from Obl/+ mutants on a C3HeB/FeJ background were outcrossed to C57BL/6J wild type females. Obl/+ F1 progeny were then backcrossed to +/+ animals from the original C3HeB/FeJ strain. Offspring from these backcross matings were examined at 2 months of age or older  using the Preyer reflex. Tail and pinna tissue were collected for DNA preparation. A total of 255 backcross mice were analysed . A genome-wide scan was conducted with 60 microsatellite markers approximately 25 cM apart, that had been shown to be polymorphic between C3HeB/FeJ and C57BL/6J inbred strains  allele, but not in the mutant (T) allele. Primers OblRTF (5′-CTT CTT CTC CCT GCC ACT GTC GTA G) and OblRTR (5′-CCA CCG AGA CAC CGG TCC CGG TTC) were used for PCR. The 111 bp PCR product was digested with StyI (New England Biolabs) which cuts the wildtype allele giving an 89 bp fragment while the mutant allele remains uncut. This genotyping tool was used to establish whether the sequence change in Obl DNA was a polymorphism in a total of 17 inbred strains: BALB/C, CBA/Ca, C3HeB/FeJ, DDY/Jc1, 129X1/SvJ, A/J, Bxd-1/Ty, C58/J, CE/J, DA/HuSn, DBA/2J, FL/1Re, LP/J, NON/LtJ, RBG/Dn, St/bJ and SWR/J.Sequence analysis was performed on genomic DNA in GCTAGCATGGCCTCCTCCGAGGACGTCA-3′5′-GC and reverse: AGATCTGAGGCGCCGGTGGAGTGGCGG-3′5′-GC, bearing restriction sites for NheI and BglII, respectively (in bold). The PCR product was then digested with NheI and BglII and inserted in pEGFP-c1  digested with NheI and BglII to create pmRFP-c1. PMCA2 w/a in pMM2  was excised by independent digestion with SalI-EcoRI and EcoRI-KpnI and inserted into XhoI-KpnI sites of pmRFP-c1 in a three-part ligation reaction resulting in pmRFP-PMCA2wa. The construct was controlled by sequencing. Site-directed mutagenesis was carried out to obtain the mutant cloned in the appropriate vector. pmRFP-PMCA2w/a was used as target and experiments were performed according to the manufacturer's standard protocol  The following primers were used: Obl5′ CATCATGGACACATTTGCTTTCCTGGCCCTGGCAACAGAGC 3′ (forward) and 5′ GCTCTGTTGCCAGGGCCAGGAAAGCAAATGTGTCCATGATG 3′ (reverse)mRFP was amplified from pCDNA3.1/zeo-mRFP  using the following primers, forward: CHO cells were grown in Ham's F12 medium, supplemented with 10% fetal calf serum (FCS). Before transfection, they were seeded onto 13 mm glass coverslips and allowed to grow to 50% confluence. Transfection with 3 µg of plasmid DNA (or 1.5 :1.5 µg in the case of co-transfection) was carried out with a Ca-phosphate procedure 2 atmosphere. Additions to the KRB medium  were made as specified in the figure legends. The experiments and luminescence calibration into [Ca2+] values were carried out according to 2+-rich solution (10 mM CaCl2 in H2O) to discharge the remaining aequorin pool. Briefly, a 13-mm round coverslip with the transfected cells was placed in a perfused thermostated chamber in close proximity to a low-noise photomultiplier, with a built-in amplifier discriminator. The output of the discriminator was captured by a Thorn-EMI photon-counting board and stored in an IBM-compatible computer for further analyses. Luminescence was calibrated off-line into [Ca2+] values by using a computer algorithm based on the Ca2+ response curve of wt aequorin. Data are reported as mean±SD. Statistical differences were evaluated by Student's 2-tailed t-test for unpaired samples. A p value<0.01 was considered statistically significant.Transfected cytAEQ were reconstituted by incubating CHO cells for 1–3 h with 5 µM coelenterazine in Dulbecco's modified Eagle's medium (D-MEM) supplemented with 1% FCS, at 37°C in a 5% CO2PO4 (anhydrous), 0.35 NaHCO3, 8.0 NaCl, 0.048 Na2HPO4 (anhydrous), 1 D-glucose. The epithelium was fixed by Cell-Tak , mixed with 90% NaHCO3, to the lateral side of a glass capillary , which had been previously glued to a microscope slide by a small drop of Sylgard Silicon Elastomer . Cultures were preserved for one day at 37°C in a complete medium of 95% D-MEM/Ham's F-12 (1∶1)  and 5% fetal bovine serum.To access utricular maculae of wild type or mutant mice between postnatal day 3 (P3) and P4, the otic capsule was opened medially and the endolymphatic compartment of the macula cut open. The otolithic membrane was removed after 15 min incubation in dissection saline to which 0.1 g/l bacterial subtilisin  had been added. Dissection saline was composed of Hank's Balanced Salt Solution  with 10 mM HEPES, 10.000 U/l penicillin and 25 µg/l fungizone. HBSS contained (in g/l): 0.4 KCl, 0.06 KHOrganotypic cultures dissected from P3 wild type and mutant mice pups were maintained over night at 37°C in D-MEM/Ham's F-12 (1∶1) medium with 5% fetal bovine serum. Tissue preparations were fixed in paraformaldehyde 4% for 20 min at room temperature, rinsed in washing solution (PBS containing BSA 2%) and permeabilised with washing solution containing Triton 0.1% for 1 h at room temperature. Incubation with primary PMCA antibody 2N was carried out overnight at 4°C using a 1∶100 dilution in washing solution. FITC-conjugated rabbit anti-IgG antibody (Invitrogen) was used as secondary antibody for pump detection . The preparation was mounted on a coverslip and imaged up side down on an inverted spinning disk confocal microscope  using a 60× oil-immersion objective at a 1.4 N.A. .Cochleae dissected from P5 mice were fixed in 4% paraformaldehyde for 20 min at room temperature, rinsed in PBS containing 2% BSA (rinse solution) and permeabilized for 1 hour at room temperature with 0.1% Triton, dissolved in rinse solution. Tissues were stained with polyclonal isoform-specific PMCA antibody 2N  by incubation overnight at 4°C with specific polyclonal antibodies (2.5 µg/ml) (Invitrogen) diluted in rinse solution. A FITC conjugated rabbit anti–IgG antibody  was used as secondary antibody, following incubation in rinse solution for 2 hours at room temperature. F–Actin was stained by incubation for 1 hour at room temperature with rhodamine phalloidin  diluted in rinse solution. Stained samples were sandwiched between two coverslips and imaged with a 60× oil–immersion objective  attached to an inverted microscope  equipped with a Nipkow disk confocal scanning head . Confocal fluorescence images were captured with a scientific grade cooled CCD camera .2 . Experiments were performed with a 100× water-immersion objective  using the same perfusion medium. Fluo-4 fluorescence was excited by the 488 nm line of an argon laser coupled by fiber optics to the confocal microscope. Fluorescence emission was selected around 528 nm using a narrow-band (50 nm) interference filter. Fluorescence images of utricle hair cells in the organotypic cultures were acquired with a resolution of 512×512 pixel by scanning at 512 lines per second under control of the Biorad Laser Sharp software. To be retained for subsequent recording, imaged cells had hair bundles extending for their entire length in a single confocal plane (bundle planarity condition), a possibility afforded by having the culture attached to a curved surface.Cultures were loaded with 10 µM cell permeant Fluo-4 AM (Invitrogen) for 50 min at 37°C in D-MEM supplemented with 10 µM cell permeant NP-EGTA AM (Invitrogen), 25 µM sulfinpyrazone and Pluronic F-127 (0.1% w/v). For de-esterification, cultures were transferred to an experimental chamber mounted on the stage of a confocal imaging setup (Biorad Radiance 2100) incorporating an upright microscope  and superfused for 20 min with a medium composed of HBSS supplemented with 4.4 g/l glucose and 2 mM anhydrous CaClF, measured in the photomultiplier tube PMT units from a value of 0 to a saturating value of 255. Laser intensity and PMT gain were adjusted to accommodate the dynamic range of changes in F. Typical background fluorescence values, measured from regions devoid of obvious cellular structures, were ∼1 PMT units, while pre-stimulus  levels, F0, averaged over the entire length of the hair bundle, were ∼8 PMT units. In all experiments pinhole aperture was adjusted to the same value, yielding confocal section with 3 µm thickness. To assay the Ca2+ extrusion activity of the PMCAs, an area of ∼3000 µm2, comprising a few hair cells in the cultured utricular macula, was exposed to UV radiation generated by an air-cooled 337 nm pulsed nitrogen laser  connected to the microscope through a 600 µm ∅ optical fiber. UV light was directed onto the sample by reflection off a 400 DCLP dichromatic beam splitter (Chroma) positioned at 45° just above the microscope objective lens. A single laser pulse (4 ns) delivering a maximum of 326 µJ of energy (at the laser output) was used to photorelease Ca2 from the caged state (NP-EGTA bound to Ca2+) in the brief time interval between the 2500th and the 2501st scan line. In a typical record lasting about 120 s, 20 000 consecutive lines were acquired.Dynamic fluorescence data were acquired in the ‘linescan’ mode to produce a scan series of fluorescence intensity values, F/F0 where ΔF = F−F0. In these expressions, raw pixel values are spatial averages along the hair bundle. Maximal percent fluorescence changes, (Fmax−F0)/F0, were about 280%. To estimate the slow time constant, τ, of recovery to baseline, transients peaking at ΔFmax, were fitted by a single exponential function during the first 10 s from the UV pulse. Data are given as mean±standard error of the mean (S.E.M.).All data were analyzed offline on a personal computer using the Matlab 7.0  software environment. Data are presented as ΔFigure S1dfw, wri, Obl), two are small deletions that lead to frame shift mutations and premature truncation of the PMCA2 protein , and one is a targeted null allele (tm1GesAtp2b2) Mutations identified to date affecting PMCA2 protein in mice. Three mutations are missense mutations leading to amino acid substitutions ((0.29 MB TIF)Click here for additional data file.Figure S2Homo sapiens), Q9ROK7 (Mus musculus), NP_036640 (Rattus norvegicus), XP_509257 (Pan troglodytes), NP_777121 (Bos taurus), NP_999517 (Sus scrofa), Q00804 (Oryctolagus cuniculus), AAK11272 (Rana catesbeiana), AAH77905 (Xenopus laevis), AAR28532 (Procambarus clarkii), AAR13013 (Stylophora pistillata), AAK68551 (Caenorhabditis elegans), XP_653525 (Entamoeba histolytica), AAP46286 (Trypanosoma brucei), EAL62716 (Dictyostelium discoideum), NP_849716 , AAB81284 (Paramecium tetraurelia), NP_001001323 (PMCA1), NP_068768 (PMCA3), NP_001675 (PMCA4), NP_004311 (SERCA1), NP_733765 (SERCA2), NP_777615 (SERCA3), AAF35375 (SPCA1), NP_000693 (Na+/K+ ATPase), and AAH31609 (Na+/K+ ATPase).Amino acid alignments of PMCA isoforms. Alignment of the amino acids residues in transmembrane domain 6 of PMCA isoforms in different species (A) and of PMCAs and other P-type ATPases (B). The similarity analysis was performed using the ClustalW program. GenBank accession numbers are listed: NP_001674 ((1.73 MB TIF)Click here for additional data file.Table S1Obl/+×+/+ matings. Progressive hearing loss detected in Obl/+ mutants starts at 1 month of age (6%), with most being deaf by 2 months of age (42%). 50% offspring from Obl/+×+/+ mating are expected to show phenotype.Progressive hearing loss in litters from (0.04 MB DOC)Click here for additional data file.Table S2Microsatellite markers polymorphic between C3HeB/FeJ and C57BL/6J.(0.17 MB DOC)Click here for additional data file.Table S3Atp2b2.Sequencing primers for (0.07 MB DOC)Click here for additional data file.Text S1Supplementary materials.(0.05 MB DOC)Click here for additional data file.


In this study we determined the level of tumour necrosis factor alpha  in liver and tumour tissue samples obtained from patients with colorectal metastases confined to the liver, who were treated with isolated liver perfusion with TNF-alpha and melphalan. We adapted a standard enzyme-linked immunosorbent assay kit for the quantification of TNF-alpha in serum to measure the amount of this cytokine in solid tissue. For this purpose, we developed a buffer that lysed the tissues without affecting the TNF-alpha present. The minimum detection level was about 2 pg of TNF-alpha per mg tissue. Using this technique, we found a significant increase in the TNF-alpha level after perfusion in the liver tissue of all evaluable patients, which may explain the transient liver toxicity we observed in all patients. In tumour tissue, a significant TNF-alpha increase was observed in one out of five patients. The level of TNF-alpha in all liver tissue samples and some of the tumours after treatment by isolated liver perfusion was much higher than the peak serum concentrations obtained after systemic administration of the maximum tolerated dose of TNF-alpha. Furthermore, we demonstrated that the level of TNF-alpha in the liver tissue samples was about seven to eight times higher than in tumour tissue. We concluded that regional liver treatment resulted in a relatively high local level of TNF-alpha, but also that this cytokine did not preferentially accumulate in tumour tissue.


Aim. The aim of this study was to determine tumor necrosis factor-alpha(TNF-Methodology. Periapical exudates were collected from root canals of 60 single-rooted teeth using absorbent paper points. TNF-Results. Nonparametric Kruskal-Wallis test revealed significant differences between TNF-Conclusions. The levels of TNF- Bacterialcell wall components react with monocytes, macrophages, other cells of the immune system, aswell as with fibroblasts, leading to the production of proinflammatorycytokines, such as IL-1Persistent injuries to the dental pulp usually cause irreversible pulpitis and pulpal necrosis. Irritants may bemechanical or chemical, but are most often bacterial. The interaction between these irritants and host defensive cells results in release of numerous mediators that, through the root canal system, are capable of initiating immunologicreactions in periapical tissues resulting in the formation on inflammatory periapical lesions. These reactions include immune response mediated by cells, through the actions of T lymphocytes and cytokines, and hummoral immuneresponse mediated by antibodies, activated by B-lymphocyte products .α is a monocyte-derived protein that has a wide range of proinflammatory and immunomodulatory effects on a number of different cell populations. TNF-α is a cytokine which stimulates boneresorption, prostaglandin synthesis, and protease production by many cell types, including fibroblasts and osteoblasts. Overproduction or inappropriate expression of TNF-α can lead to a variety of pathological conditions [2, IL1-β, and TNF-α has been demonstrated in periapical lesions [TNF-nditions . The loc lesions , 5.α is a cytokine initiallyidentified as a causative of hemorrhagic necrosis in certain tumors and waslater shown to be the same molecule as cachectin, a serum product earlier knownas a mediator of wasting in chronic disease. In addition to its diversebioactivities, TNF-α is the only molecule other than IL-1 that is presentlyknown to have osteoclast-activating function. Although extensive research hasbeen performed in the area of periapical inflammatory mediators, only few studiesfocused on the role and levels of TNF-α in periapical exudate [α in periapical exudates andto evaluate possible relationships between this cytokine and radiologicalfindings in the involved teeth.TNF- exudate , 7. The The investigation was carried out on 60 subjects of different age andsex. Subjects were divided into three groups according to the radiologicalfindings. Diagnoses were established according to the clinical and radiologicalfindings. All patients in the certain period were included. The patients withnoncontributory medical histories and diagnosed with apical periodontitis wereselected for the study as follows.Group 1 consisted of 20 subjectswhose single-rooted teeth had the diagnosis of chronic apical periodontitis,with dull or absent pain, and size of the lesion <1 cm.Group 2 consisted of 20 subjectswhose single-rooted teeth were diagnosed with chronic apical periodontitis,with dull or absent pain, but with the lesion size of >1 cm.Group 3 was control andcomprised of 20 subjects whose investigated single-rooted teeth showed symptomsof acute periapical condition accompanied by excruciating pain, necrotic pulp,and radiological thickening of the apical periodontal ligament.Primary access to the pulp chamber was gained using slow-speed round dental burs, withplacement of rubber dam isolation. After determination of the working length, thenecrotic pulp was removed and the root canal was enlarged to ISO size 40. Theroot canal was dried with sterilized paper points. A sterile paper point wasinserted into the root canal up to the instrumentation length and held in placefor 1 minute to absorb the exudate for the sample [e sample , 7. Papeanalysis , 9. The α levels in supernatant of periapical exudate isbased on utilization of monoclonal antibodies specific for TNF-α which bindcompetitively to TNF-α. Investigation is based on “sandwich” enzymeimmunoassay (EIA).Determination of the TNF-All subjects were informed of the aims and procedures of research. Withinthe research, they are guaranteed in respect of their basic ethical and bioethical principles:personal integrity  asregulated by Nαin all periapical exudate samples. Differences in concentrations of TNF-α betweencontrol group , the group with large radiolucent areas, as well as between control and the canals associated withsmall radiolucent areas  were statistically significant (P < .05).There was also statistically significant difference between the samples withlarge radiolucent areas and small radiolucent areas. EIA data for TNF-α werecategorized according to radiological diagnosis, using the nonparametricKruskal-Wallis test , and matrix metalloproteinases (MMP), which are in charge ofdestroying extracellular matrix of the bone tissue [It was proved that TNF-e tissue .α-positive cells within the granuloma with a macrophage-likemorphology. Ultrastructural analyses showed that there are some macrophages which have adjusted the extracellularsecretion; therefore, these macrophages might be the mainsource of cytokines in the tissue.The work by Artese et al.  showed tα and IL-1 are secreted inthe infected rat pulps and periapical lesions. The cells appeared as soon as 2days following the pulp chamber opening and their numbers increased steadilyuntil the day 30. These findings demonstrate the presence of IL-1α- and TNF-α-secreting cells in the pulpand periapical tissues immediately following the pulp exposure, which supportsthe assumption that the abovementioned cytokines play a role in pathogenesis ofpulp and periapical pathoses.Wang et al.  showed tα levels in healthy, symptomatic, and asymptomatichuman pulps [α was detected in all vital pulp tissues. The highestconcentrations of TNF-α, with regard to clinical classification, were found insymptomatic reversible pulpitis, and the difference was statisticallysignificant in comparison with irreversible asymptomatic pulpitis and healthypulp. The highest concentrations of TNF-α, with regard to histologicalclassification, were found in histologically confirmed moderate inflammation,followed by severe inflammation, mild inflammation, and finally healthy pulp.In the analysis of TNF-an pulps , TNF-α wα could be an objective markerin radiologically confirmed periapical lesions. Presence of TNF-α in thisinvestigation was proved in all clinical samples. The lowest levels weredemonstrated in control group, which was characterized by radiologicalthickening of the periodontal ligament, while somewhat greater concentrationswere found in teeth with periapical radiolucencies of <1 cm diameter; finally,the greatest concentrations were measured in teeth with radiolucent lesions>1 cm, which confirms the relationship between TNF-α and the emergence anddevelopment of periapical lesions. Objective analysis of TNF-α levels enablesestablishment of a relationship between different concentrations of thiscytokine and different radiological changes.Conclusion can be drawn that cytokine TNF-


An isolated limb perfusion (ILP) model using soft tissue sarcoma-bearing rats was used to study prerequisites for an effective ILP, such as oxygenation of the perfusate, temperature of the limb, duration of the perfusion and concentration of tumour necrosis factor (TNF). Combination of 50 μg TNF and 40 μg melphalan demonstrated synergistic activity leading to a partial and complete response rate of 71%. In comparison to oxygenated ILP, hypoxia was shown to enhance anti-tumour activity of melphalan alone and TNF alone but not of their combined use. Shorter perfusion times decreased anti-tumour responses. At a temperature of 24–26°C, anti-tumour effects were lost, whereas temperatures of 38–39°C or 42–43°C resulted in higher response rates. However, at 42–43°C, local toxicity impaired limb function dramatically. Synergy between TNF and melphalan was lost at a dose of TNF below 10 μg in 5 ml perfusate. We conclude that the combination of TNF and melphalan has strong synergistic anti-tumour effects in our model, just as in the clinical setting. Hypoxia enhanced activity of melphalan and TNF alone but not the efficacy of their combined use. For an optimal ILP, minimal perfusion time of 30 min and minimal temperature of 38°C was mandatory. Moreover, the dose of TNF could be lowered to 10 μg per 5 ml perfusate, which might allow the use of TNF in less leakage-free or less inert perfusion settings. © 1999 Cancer Research Campaign


Survival analysis has found widespread applications in medicine in the last 10-15 years. However, there has been no published review of the use and presentation of survival analyses. We have carried out a systematic review of the research papers published between October and December 1991 in five clinical oncology journals. A total of 132 papers were reviewed. We looked at several aspects of study design, data handling, analysis and presentation of the results. We found that almost half of the papers did not give any summary of length of follow-up; that in 62% of papers at least one end point was not clearly defined; and that both logrank and multivariate analyses were frequently reported at most only as P-values [63/84 (75%) and 22/47 (47%) respectively]. Furthermore, although many studies were small, uncertainty of the estimates was rarely indicated [in 13/84 (15%) logrank and 16/47 (34%) multivariate results]. The procedure for categorisation of continuous variables in logrank analyses was explained in only 8/49 (16%) papers. The quality of graphs was felt to be poor in 43/117 (37%) papers which included at least one survival curve. To address some of the presentational inadequacies found in this review we include new suggested guidelines for the presentation of survival analyses in medical journals. These would complement the statistical guidelines recommended by several clinical oncology journals.


This study was conducted to increase the anti-tumour potency and reduce the toxic side-effects of tumour necrosis factor alpha . Natural human TNF-alpha was chemically conjugated with monomethoxy polyethylene glycol (PEG) using succinimidyl coupling of lysine amino groups of TNF-alpha. The number-average molecular weight of PEG-modified TNF-alpha  increased with an increase in the reaction time and the initial molar ratio of PEG relative to TNF-alpha. The resulting modified TNF-alpha was separated into fractions of various molecular weights. The specific activity of separated PEG-TNF-alpha s relative to that of native TNF-alpha gradually decreased with an increase in the degree of PEG modification, but the plasma half-life was drastically increased with the increase in molecular weight of modified TNF-alpha. PEG-TNF-alpha s, in which 29% and 56% of lysine residues were coupled to PEG, had anti-tumour activity approximately 4 and 100 times greater than unmodified TNF-alpha in the murine Meth-A fibrosarcoma model. Extensive PEG modification did not increase its in vivo activity. A high dose of unmodified TNF-alpha induced toxic side-effects, but these were not observed with the modified TNF-alpha s. Optimal PEG modification of TNF-alpha markedly increased its bioavailability and may facilitate its potential anti-tumour therapeutic use.


In this study, we extend these investigations asking specifically whether the TNF-α effect on cell invasion and migration involves activation of proteolytic enzymes. We examined the effect of TNF-α on melanoma expression/activation of type IV gelatinases matrix metalloproteinases 2 and 9 (MMPs -2 and -9) and general proteolytic enzymes. Stimulation with TNF-α significantly increased both melanoma cell migration at 24 h (+21%) and invasion through fibronectin (+35%) but did not upregulate/activate the expression of latent MMP-2 constitutively produced by these cells and did not upregulate their general protease activity. However, the increased cell migration and invasion through fibronectin observed following stimulation with TNF-α were inhibited by the general protease inhibitor α2 macroglobulin. These findings suggest that the promigratory and proinvasive effect of TNF-α on this melanoma cell line may be mediated to some extent by induction of localised cell membrane-bound degradative enzyme activity, which is not readily detected in biochemical assays.Inflammatory mediators have been reported to promote malignant cell growth, invasion and metastatic potential. More specifically, we have recently reported that tumour necrosis factor alpha (TNF- Proteolytic degradation of the basement membrane and the extracellular matrix (ECM) and movement through it are regarded as essential steps in the cascade of events leading to successful tumour cell invasion and metastasis. Studies on human melanoma have provided evidence that the plasminogen activator system and the matrix metalloproteinase (MMP) enzyme systems play an important role  opposed cell attachment to ECM substrates and cell invasion through fibronectin, both of which would be consistent with TNF-α promoting metastasis and α-MSH providing partial protection against the action of TNF-α.Studies on the effects of TNF-α on melanoma cell migration and degradative enzyme activity.In the current study, we extend our investigations into the role of inflammation in melanoma invasion by studying the effects of TNF-α on melanoma cell general proteolytic enzyme expression and expression/activation of MMPs -2 and -9 and studied the extent to which degradative enzymes are involved in the TNF-α induced cell responses by looking at the effect of the general protease inhibitor α2 macroglobulin on TNF-α stimulated cell migration and invasion.Specifically, we examined the effect of TNF-α and α2 macroglobulin from Roche Diagnostics (Germany); and fluorescent BODIPY® casein (EnzChek® Protease Assay Kit-green fluorescence) from Molecular Probes Inc. . Tissue culture plastics were purchased from Corning Costar Corporation . All other chemicals were of analytical grade.Culture medium, additives and antibiotics were obtained from Gibco BRL  and Sigma Chemicals Ltd. . Foetal calf serum (FCS) was purchased from GlobePharm Limited ; newborn calf serum (NBCS) from Sigma Chemicals Ltd. ; trypsin/ethylenediaminetetraacetic acid (EDTA) and phosphate-buffered saline (PBS) tablets from Oxoid Ltd. . Human fibronectin was purchased from Sigma Chemicals Ltd. ; human recombinant TNF-−3 mol l−1 glutamine, 100 IU ml−1 penicillin and 100 μg ml−1 streptomycin sulphate.The human cutaneous melanoma cell line HBL was established in Professor Ghanem's laboratory from a lymph node metastasis of a nodular malignant melanoma , 0.2% (w/v) D-glucose solution, 0.1% (w/v) bovine serum albumin (BSA), 2 × 10−3 mol l−1 glutamine, 100 IU ml−1 penicillin, 100 μg ml−1 streptomycin sulphate and the relevant concentration of TNF-α was added. This serum-free medium was used for the fibronectin invasion assays as described later (serum-free invasion assay medium – SFIAM) and subsequently in all the assays where a 24-h preincubation period was required in order to keep the experimental culture conditions the same.For the experiments where a 24-h preincubation was required, the cell culture medium was changed to RPMI-1640 medium (Gibco) supplemented with 20 ng mlα and α2 macroglobulin on the invasion of the melanoma cells through a layer of human fibronectin. Briefly, a suspension of 1.8 × 105 HBL melanoma cells (contained in 500 μl SFIAM with or without the appropriate drug concentration as required) was added to Nunc™ tissue culture inserts containing a polycarbonate filter with 8 μm diameter pores covered with fibronectin (100 μg ml−1). Cells were incubated for 20 h under standard culture conditions. At the end of the incubation period, the cells that had invaded through the filter (cells attached to the bottom of the well and to the undersurface of the filter) were collected separately from the cells that had not invaded (cells remaining attached to the upper surface of the filter). A combined analysis of the cell counts was used to calculate the percentage of the total population of cells that had invaded through the fibronectin monolayer.A modification of the method we first described in α 200 U ml−1 for the last 24 h before passaging and adding to the Nunc™ tissue culture inserts.When a preincubation period was required prior to the invasion assay, the cells were cultured in SFIAM with or without TNF-α2 macroglobulin was used as a general protease inhibitor after initially confirming its activity against trypsin prior to using it to inhibit proteolytic activity in the cell culture medium during the fibronectin invasion assay.α-stimulated cells (incubated with TNF-α 200 U ml−1 for 24 h) were detached from the tissue culture flask and were lysed using cell extraction buffer  for 15 min at 4°C. The cell conditioned medium was also collected for gelatin zymography analysis (following centrifugation at 2000 g for 5 min to remove any cellular debris).Cell extraction of MMPs was performed when cell cultures were 70–80% confluent. Control and TNF-3 cells per well with or without TNF-α  for 24 h. At the end of the preincubation period, the conditioned medium was collected for measurement of proteolytic activity, and extraction from the cells was performed according to the protocol described above for extraction of MMPs.For the purpose of extraction of general proteolytic activity, the melanoma cells were cultured in a 24-well plate at a density of 50 × 10s-indacene-3-propionic acid (BODIPY® FL) . BODIPY® FL casein is a conjugate consisting of casein labelled with the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-μg ml−1 was used in each experiment to demonstrate proteolytic degradation of BODIPY® casein. The above concentration was selected after initial experiments to confirm that the fluorescence emitted by the cleaved substrate was proportional to the concentration of trypsin. The following controls were also included in each assay: BODIPY® casein alone, fresh culture medium (SFIAM) with BODIPY® casein and extraction buffer with BODIPY® casein. Cell extracts were diluted 1 : 20 in assay buffer before being added to the fluorescent substrate as experimentation showed this to be the optimum dilution to minimise the effect of the extraction buffer itself on the fluorescent BODIPY® substrate.As an internal control for the assay, trypsin at a concentration of 3.3 α treated cell extracts (24-h cell preincubation in SFIAM containing TNF-α 200 U ml−1 as described above) and conditioned medium (collected at the end of the 24-h preincubation period). Proteins were separated in nonreducing, nondenaturing conditions using a 10% resolving polyacrylamide gel containing 0.5% gelatin substrate. Internal human purified MMP-2 and MMP-9 standards  were included at concentrations of 20 ng per lane. After electrophoresis, the zymographs were developed, fixed and stained according to the protocol described by Zymography was carried out on control (nontreated) and TNF-A modified ‘scratch wound’ migration assay was used for the assessment of the migration of the melanoma cells. The method was previously described by 5 cells per ml per well and were incubated for 24 h under standard culture conditions. After 24 h, when the cells had attached to the bottom of the tissue culture dish, the confluent cell monolayer was scratched with a plastic pipette tip to create a cell free zone in the well. The culture medium was removed and was replaced with an equal volume of fresh SFIAM with or without TNF-α 200 U ml−1 or α2 macroglobulin. The cells were incubated for a further 24 h under standard culture conditions. During this period, photographs of the scratched cell monolayer were taken at predetermined time points  using an Apple McIntosh video microscope (Open Lab 3.0.4 software) and measurements of the distance between the two edges of the scratch were recorded for each different time point. The percentage reduction of the distance between the scratch edges at the different time points represented the migration rate of the melanoma cells. Triplicate wells were used for each experimental condition.Cells were plated in a 24-well culture plate in serum containing Ham's F10 culture medium at a density of 2 × 10vs stimulated cell samples were analysed using Student's paired t-test for fibronectin invasion assays, migration assays and for general protease activity assays.Control α ranging from 100 to 500 U ml−1 on HBL melanoma cell invasion through fibronectin over 20 h. Unstimulated (control) cells showed a mean invasion rate of 24.6±2.7% (n=4 experiments). Stimulation with TNF-α 100 U ml−1 significantly increased the invasion rate of the cells to 33.3±3.5% (P<0.05), which represents an increase of +35% above the control level of invasion. Stimulation with higher concentrations of TNF-α (250 and 500 U ml−1) was less effective and the increases in invasion did not reach statistical significance.Figure 1Aα, cells were preincubated with TNF-α for 24 h prior to the invasion assay and were also subsequently stimulated with TNF-α again during the period of the invasion assay (a further 20 h). Preincubation with TNF-α resulted in a 43% increase in invasion above control level . Further addition of TNF-α to the invasion assay did not increase any further the invasiveness of these preincubated cells (results not shown). On the basis of these data, we concluded that the biological effects of TNF-α on invasion had occurred fully within 24 h.To determine the optimum period of exposure for maximum response to TNF-α on melanoma cell migration. Cells stimulated with TNF-α 200 U ml−1 (cytokine present during the 24-h assay) had a significantly higher migration rate compared to the control (nonexposed to TNF-α) cells. The migration rate (represented by the percent reduction of the scratch width) of the TNF-α stimulated cells was 53.2±4.4% by 24 h, whereas the migration rate of the control cells at the same time point was 43.8±2.9%. This represented a 21% increase in migration as a result of TNF-α stimulation, P<0.05 based on eight experiments (as illustrated in α stimulated) at 8 or 24 h.A ‘scratch wound’ model was used to examine the effect of TNF-α 200 U ml−1for 24 h prior to cell extraction and collection of cell conditioned medium for gelatin zymography . α2 macroglobulin at a concentration of 2 U ml−1 was used for future experiments as this achieved 84% inhibition of trypsin activity.To confirm that the fluorescence emitted by cleavage of the BODIPY® casein substrate was representative of proteolytic enzyme activity, initial experiments used serial dilutions of trypsin  and the lower chamber (invading cells) was sampled at the end of the 20-h assay period and the fluorescence of the sample, representing protease activity, was measured. TNF-α did not increase general protease activity in the conditioned media in these experiments (results not shown).To measure noninvasively whether cell surface bound proteolytic activity was modified by TNF-α on proteolytic activity of HBL melanoma cells, it was still conceivable that levels may have been beneath the level of detection of these biochemical assays and that proteases played some role in the invasion of these cells through fibronectin. Accordingly, we used α2 macroglobulin, an inhibitor of all classes of endoproteases, to examine its effect on control and TNF-α stimulated HBL melanoma cells. α2 macroglobulin inhibits proteases present in the cell conditioned medium or on the cell surface, but it cannot enter the cell or penetrate rapidly into the extracellular matrix due to its large size (720 kDa tetrameric glycoprotein).Although we had failed to show any effect of TNF-α2 macroglobulin (2 U ml−1) was added to the cell suspension in the upper chamber with or without TNF-α 200 U ml−1 for the 20-h culture period of the assay. Controls included cells with no stimulants and cells where only TNF-α was added during the invasion assay. In three separate experiments, α2 macroglobulin had no significant effect on cell invasion through fibronectin (−6% of the control level of invasion), as summarised in Figure 4AP<0.05) in invasion observed following stimulation with TNF-α (+21% of the control) was completely inhibited by the addition of α2 macroglobulin.Using the model of fibronectin invasion, α2 macroglobulin (2 U ml−1) was added to cells alone or in combination with TNF-α (200 U ml−1) for the 24-h duration of the migration assay. The migration rate of the non-TNF-α stimulated cells decreased by −26%  when α2 macroglobulin was added  was completely inhibited by the addition of the protease inhibitor  insults. As such, it is invariably associated with the early phases of wound healing and persists to a certain degree, until regeneration of the injured tissues and restoration of function have been achieved. .vs narrow margin excisions were compared  and significant inhibition by α-MSH in all the cell types examined  within 24 h of TNF-α exposure for the HBL melanoma cells  expression by the malignant cells . Upregulα stimulation, we examined the expression and activation of MMPs -2 and -9 using gelatin zymography, and the general protease activity of the melanoma cells using a quenched fluorescent substrate assay. As maximal upregulation of cell invasion was achieved with TNF-α between 100 and 250 U ml−1 and previous data from our group demonstrated maximal activation of NFκB in these cells with 200 U ml−1 of TNF-α  in SK-MEL-109 melanoma cells. This suggests that TNF-α may be a potent regulator of degradative enzyme activity during malignant cell invasion. More importantly, this study demonstrates that the TNF-α cell response can be modulated (both upregulated and downregulated) at a postreceptor level via activation of different second-messenger systems suggesting that the effect of TNF-α on various target cells can be modified inside the cell to produce the cell's final response to the cytokine.The literature also reports that TNF-α on the functional biology of different human malignancies including melanoma (Although our current study reports observations made on a single cell line, our data are in agreement with previous reports from the literature on the effects of TNF-melanoma  and exteα upregulates malignant melanoma invasion and migration in vitro. Taken together, the results of our current study combined with the data we have previously published (α may exert its proinvasive effect on HBL melanoma cells via an integrin-dependent mechanism as well as a modest upregulation of degradative enzyme activity not readily detected in general protease assays.In conclusion, we report that the proinflammatory cytokine TNF-


Sera from rabbits injected with BCG and then with endotoxin contain a factor (tumour-necrosis factor TNF) which, even at high dilutions, is cytotoxic in vitro for mouse L cells and some other cell lines. Using a 51Cr-release assay, cytotoxicity was detected as early as 7-8 h after addition of TNF serum to L cells and cell death was evident microscopically by 24 h. TNF was cytotoxic at 37 degrees C but not at 21 degrees C or 4 degrees C, and acted on both dividing and non-dividing cells. The antimetabolites sodium azide and dinitrophenol partially protected L cells from TNF, suggesting that actively metabolizing cells are the most sensitive. Treatment of L cells with trypsin did not delay cytotoxicity nor was cytotoxicity inhibited in the presence of various saccharide derivatives of cell-surface glycoproteins. Rabbit TNF was remarkably stable with a mol. wt. of 40-50,000. It was eluted with the more acidic serum proteins on ion-exchange chromatography, but precipitated in 50%-saturated ammonium sulphate. Sensitivity to TNF could not be correlated with tumourigenicity of several animal and human lines tested nor with the production of C-type viruses.


During infection with human immunodeficiency virus (HIV), immune pressure from cytotoxic T-lymphocytes (CTLs) selects for viral mutants that confer escape from CTL recognition. These escape variants can be transmitted between individuals where, depending upon their cost to viral fitness and the CTL responses made by the recipient, they may revert. The rates of within-host evolution and their concordant impact upon the rate of spread of escape mutants at the population level are uncertain. Here we present a mathematical model of within-host evolution of escape mutants, transmission of these variants between hosts and subsequent reversion in new hosts. The model is an extension of the well-known SI model of disease transmission and includes three further parameters that describe host immunogenetic heterogeneity and rates of within host viral evolution. We use the model to explain why some escape mutants appear to have stable prevalence whilst others are spreading through the population. Further, we use it to compare diverse datasets on CTL escape, highlighting where different sources agree or disagree on within-host evolutionary rates. The several dozen CTL epitopes we survey from HIV-1 gag, RT and nef reveal a relatively sedate rate of evolution with average rates of escape measured in years and reversion in decades. For many epitopes in HIV, occasional rapid within-host evolution is not reflected in fast evolution at the population level. HIV evolves so quickly that it can be seen to adapt within one infected person. Evolutionary escape from immunity is particularly well-described. Escape variants transmit to new hosts, where they may revert. We present a mathematical model of three processes: within-host evolution of escape mutants, transmission of those variants between hosts and subsequent reversion in new hosts. Using this model we reconcile diverse datasets on HIV immune escape, highlighting where multiple data sources agree or disagree on the underlying rate processes. The several-dozen immune epitopes we survey reveal a relatively sedate rate of evolution with average rates of escape measured in years and reversion in decades. Although there are frequent reports in the literature of early and rapid within-host evolution of HIV, for many epitopes this is not reflected in fast evolution at the population level. During the course of a single infection HIV evolves to escape from the selection pressures imposed by its host's immune response. Such changes have been recorded under selection from all three arms of the specific immune response, but escape from CD8+ cytotoxic T lymphocytes is particularly well documented Different hosts make immune responses to different parts of HIV (known as epitopes) and for CTL responses the epitopes that can be recognised are determined by the host's class 1 human leukocyte antigen (HLA) type. CTL escape mutants can revert to the wild-type when they are no longer under selection pressure from host immune responses A large literature describes the evolution of HIV CTL escape mutants within individual hosts. Many of those papers are case reports of the timing and speed of outgrowth of escape mutations within an individual and in most cases the events described occur during the first year of infection A better understanding of the global tempo of antigenic change in HIV can be achieved by addressing a series of specific questions. On average, how fast do HIV escape mutations arise in HLA matched individuals? How fast do reversions occur in HLA mismatched people? HIV is a relatively recently emerged infection of humans; so is it still adapting to its new hosts, and if so, how fast? What is the relationship between the tempo of adaptation within individuals and the rate of genetic change across the entire pandemic? If HIV is still adapting, what patterns can we expect to unfold across the population of infected people? How will those patterns be different in people of different HLA types and in populations with different HLA frequencies?Some of these questions have been elegantly addressed in large observational studies which describe the patterns of events that have unfolded in recent decades In order to address this gap we have developed a mathematical model that simultaneously captures events while viruses evolve within individuals and tracks the spread of variants as viruses are transmitted between individuals. Between-host transmission is modelled using a standard mathematical description of the frequency-dependent transmission of an infectious disease from which there is no recovery – the so-called SI model. However, the model we present allows host-heterogeneity with respect to a single HLA type so that some hosts have the potential to make a CTL response to a given viral epitope, whereas other hosts do not. Viral evolution is captured by allowing viral heterogeneity with respect to the presence – or not – of escape mutations in a single epitope restricted by the host HLA under consideration. Thus there are four types of infected hosts: HLA matched hosts infected with wild-type or escape mutant virus, and HLA mismatched hosts infected with wild-type or escape mutant virus. Thus there are no mixed infections, or more precisely each host can only be infectious with one type of virus. Only HLA matched hosts infected with wild-type virus can mount effective CTL responses to the viral epitope under consideration. They drive the evolution of CTL escape mutants and can therefore switch to become HLA matched hosts infected with escape mutant virus. HLA mismatched hosts are unable to mount CTL responses to the given epitope whatever mutations it bears and they can therefore allow their infecting virus to revert from escape mutant to wild-type. In this model such viral reversion is represented by HLA mismatched hosts switching from being infected with the escape mutant virus to being infected with the wild-type virus. Every infected host is infectious with the viral type they carry, so that the two viral types are transmitted between individuals at rates driven by the proportion of the total population infected with each. A diagram of the model is presented in This model has similarities to mathematical models of the spread of drug resistance In each of In any mutation at a site at which an escape mutation has been described . Escape data are available at two levels of organisation: comparisons across individuals (www.hiv.lanl.gov) using a search for dated B-clade sequences and eliminating duplicate samples from the same individual. Although this database is not strictly an epidemiological survey, it is the largest source of temporal population level data. The six epitopes are a subset of 31 epitopes in gag, reverse transcriptase (RT), and nef for which at least one escape mutation has been described in the literature. Details and references for these mutations are provided in http://hivdb.stanford.edu/). As predicted in The model's behaviour can be compared with CTL escape data from the current HIV pandemic. Such data are available from diverse studies, summarised in ividuals  and chanividuals . Figure R0, , the average life expectancy of infected hosts, the duration of the HIV epidemic in the sample population and the proportion of the population who are HLA matched for each epitope. Since the data in 2B are from Switzerland, we assume that HLA prevalences are equal to those found in Caucasians We do not have to wait several decades for longitudinal cohort studies to play out. Our model can be used to infer rates of escape and reversion from HLA-typed escape prevalence data such as that shown in To test our model predictions, escape and reversion rates for the same epitopes were determined from the independent longitudinal cohort data (dataset 4) presented in We can take the inferred rates of escape and reversion and use the model to calculate a predicted change over 20 years (1985–2005) in the population escape prevalence of each epitope. When we compare that predicted change with the observed change in sequences deposited in the Los Alamos database  and S3 wOur interpretation of the data in Our inferred reversion rates are even slower than our inferred escape rates: there is no reversion in 56% of epitopes and at the lower quartile the average time to reversion is 6.5 years. As shown in It is surprising to find that the rate of escape from CTL selection is more than an order of magnitude slower than suggested in a substantial literature describing events in carefully followed individuals. It is reassuring that the result arises very consistently from two different analyses of two independent datasets. Estimating escape and reversion rates from longitudinal cohort data is a straightforward and well-established process see  legend. in vitro to confer escape. This curbs both the epitopes we look at and the sites within those epitopes that we consider. To check for bias arising from only looking at sites with defined escape we replot Might our definition of escape have excluded many genuine escape mutations? We confined our analysis to mutations at sites that have been demonstrated It is an inbuilt assumption of our model that escape mutants do not revert in HLA matched hosts. In principal such reversion could lead to lower prevalence of escape in HLA matched hosts and consequent underestimation of escape rates. The longitudinal cohort study (dataset 4) allows an estimate of the rate at which escape mutants revert in HLA matched hosts. The estimate is that this occurs, on average, once every 25 person-years of observation. Using this estimate and a modified version of our model in which escape mutants can revert in HLA matched hosts we can re-estimate escape and reversion rates from the data in Another possibility is that some escape mutations only appear transiently, to be replaced by other mutations in the same epitope. We develop a model of such a process in We have assumed that the rates of escape and reversion are homogenous across the duration of infection. In principal, if escape is faster earlier on during infection the prevalence of escape mutants in the population would be lower than would be predicted under the assumption that escape is as fast late on as it is early on during infection. This would lead us to underestimate the rate of escape during the early stage of infection. Instead, our estimate would represent a form of average escape rate across both the early and late stages of infection. Likewise, faster reversion in early infection could affect the prevalence of escape mutants and thus our inferred rates. Using a model in which escape and reversion are both faster in the first year of infection and an estimate  that both rates halve after the first year we re-analysed the cross-sectional data to see how estimates change under these different assumption. We found that both inferred escape and reversion rates are faster under these new assumption but the halving of rates after 1 year is not large enough to have a substantial effect upon the escape prevalence at the population level and thus upon our inferred rates of escape and reversion. Details of the model and of the data supporting a halving of rates after one year are presented in We have also assumed that people are equally infectious throughout their infection. A study of HIV-discordant heterosexual couples Throughout this analysis we have treated different epitopes as though they were independent entities. Of course that is not so and the most intimate way in which epitopes can interact is by lying in identical parts of the HIV genome. Many CTL epitopes do lie in overlapping sections of the genome and we therefore developed a model to investigate the dynamics of a mutation at a single site that confers resistance in two overlapping epitopes restricted by two different HLA alleles . All patients provided written informed consent before participating in this study.t = timeh = host type v = virus type (0 if wild-type (WT), 1 if escape mutant (EM))B = population birth rate (years−1)p = proportion of the population who are HLA matched−1)ψ = rate of reversion in HLA mismatched hosts (years−1)μ = death rate of susceptible hosts (years−1)α = disease-related death rate (years−1)β = transmission probability per partnershipc = rate of partner exchange (years−1)h(t)X = number of susceptible hosts of host type h at time th hosts infected with virus type v at time tN(t) = total number of hosts in the population at time tv(t)λ = force of infection from hosts infected with virus type v at time t (years−1)The model is described mathematically using ordinary differential equations (1–6), where the force of infection is defined as Susceptible, HLA mismatchedSusceptible, HLA matchedInfected, WT, HLA mismatchedInfected, EM, HLA mismatchedInfected, WT, HLA matchedInfected, EM, HLA matchedMinitab 14 was used to compare the observed and predicted escape rates . Firstlyvalences  were comon rates  could noTable S1A summary of mutations in HIV-1 gag, RT and nef which have been reported in the literature as conferring CTL escape and confirmed by in vitro tests.(0.02 MB PDF)Click here for additional data file.Table S2A summary of escape and reversion data from the cross-sectional study (dataset 2) and the longitudinal cohort study (dataset 4).(0.03 MB PDF)Click here for additional data file.Table S3A summary of published escape data from case reports (dataset 3).(0.03 MB PDF)Click here for additional data file.Table S4A summary of published reversion data from case reports (dataset 3).(0.01 MB PDF)Click here for additional data file.Text S1Analytic expressions representing the escape prevalence under different circumstances.(0.03 MB PDF)Click here for additional data file.Text S2Supporting references.(0.01 MB PDF)Click here for additional data file.Text S3A detailed analysis describing how additional factors could affect the evolution of escape mutants and thus affect our inferred rates of escape and reversion rates from the cross-sectional data (dataset 2).(0.41 MB PDF)Click here for additional data file.Figure S1Confidence intervals for escape and reversion rates inferred from the cross-sectional data (dataset 2).(0.04 MB PDF)Click here for additional data file.Figure S2Sensitivity analysis showing how the assumed model parameters affect our inferences from the cross-sectional data c.f. .(0.19 MB PDF)Click here for additional data file.Figure S3Observed changes in the population escape prevalence over approximately 20 years (dataset 1).(0.04 MB PDF)Click here for additional data file.


The vaccine components were administered intramuscularly to BALB/c mice either separately or in the trivalent combination. High levels of NoV and RV type specific serum IgGs with high avidity (>50%) as well as intestinal IgGs were detected in the immunized mice. Cross-reactive IgG antibodies were also elicited against heterologous NoV VLPs not used for immunization  and to different RVs from cell cultures. NoV-specific serum antibodies blocked binding of homologous and heterologous VLPs to the putative receptors, histo-blood group antigens, suggesting broad NoV neutralizing activity of the sera. Mucosal antibodies of mice immunized with the trivalent combination vaccine inhibited RV infection in vitro. In addition, cross-reactive T cell immune responses to NoV and RV-specific antigens were detected. All the responses were sustained for up to six months. No mutual inhibition of the components in the trivalent vaccine combination was observed. In conclusion, the NoV GI and GII VLPs combination induced broader cross-reactive and potentially neutralizing immune responses than either of the VLPs alone. Therefore, trivalent vaccine might induce protective immune responses to the vast majority of circulating NoV and RV genotypes.Rotavirus (RV) and norovirus (NoV) are the two major causes of viral gastroenteritis (GE) in children worldwide. We have developed an injectable vaccine design to prevent infection or GE induced with these enteric viruses. The trivalent combination vaccine consists of NoV capsid (VP1) derived virus-like particles (VLPs) of GI-3 and GII-4 representing the two major NoV genogroups and tubular RV recombinant VP6 (rVP6), the most conserved and abundant RV protein. Each component was produced in insect cells by a recombinant baculovirus expression system and combined  Acute gastroenteritis (AGE) is a leading cause of morbidity and mortality in children all over the world . Virusesin vitro [Development of a NoV vaccine is underway –8. Sincein vitro . An addiin vitro . The majin vitro . There iin vitro . For ovein vitro ,14. Therin vitro  but no pin vitro . It has in vitro –19.RV annually accounts for ~450 000 deaths in children under 5 years of age, the majority of the deaths taking place in developing countries . Since tin vitro [RV has a double stranded RNA genome enclosed in the triple layered capsid . VP7 forin vitro  and thesin vitro .We have previously shown that a dual combination of NoV GII-4 VLPs and RV rVP6 tubules induced strong humoral immune responses without mutual inhibition when delivered parenterally into BALB/c mice . In the The protocol for the study was approved by the Finnish National Animal Experiment Board (permission number ESLH-2009-06698/Ym-23). All the procedures performed on the animals were conducted according to the guidelines of the Finnish National Animal Experiment Board and all efforts were made to minimize animal suffering.in vitro antigenicity and protein concentration were determined for each protein as described previously [NoV GII-4 VLPs, GI-3 VLPs, GII-4 New Orleans (NO) VLPs, GII-12 VLPs, GI-1 VLPs, and RV rVP6 used for immunizations and/or as antigens in immunological assays were produced by a baculovirus-insect cell expression system and purified by sucrose gradients as previously described ,36. The eviously ,36.The RV cultures used as antigens in the enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunosorbent spot (ELISPOT)-interferon-γ (IFN-γ) assays were propagated in an MA104 cell line  as described by others ), BrB  of NoV GII-4 VLPs, GI-3 VLPs or RV rVP6 were administered intramuscularly (IM) to 7-week-old female BALB/c mice . The mice were immunized (5 mice/group) at study weeks 0 and 3 and euthanized at study week 5. After the optimal dose selection, naïve BALB/c mice (5 mice/group) were immunized in another set of experiments according to the above schedule with a single NoV GII-4 VLPs, GI-3 VLPs or RV rVP6 antigen (each at a 10 µg dose) or the trivalent combination (10 µg GII-4 VLPs + 10 µg GI-3 VLPs + 10 µg rVP6) and euthanized at study week 5. A group of mice receiving the trivalent combination vaccine (7 mice/group) were euthanized at study week 27 for the long-term follow-up of the immune responses. Negative control groups of mice (5-7 mice/group) received carrier only (PBS) and were terminated at week 5 or week 27. Blood samples were collected at study weeks 0 (pre-immune serum), 3, 4, 7, 12, 16, and 20 as previously described . Whole bELISA used to measure antigen-specific IgG, IgG1, and IgG2a from serum is described in details elsewhere ,38. BrieSerum IgG avidity was measured by ELISA as described above with an extra urea incubation step to remove the low avidity antibodies ,40. The NoV-specific IgG content was tested from stool suspensions (10% suspension) with the ELISA as described above. RV rVP6-specific IgG and IgA in the stool suspensions and VWs and rVP6-specific IgA in serum were detected by sandwich ELISA as follows. The 96-well plate was first coated with rabbit anti-rotavirus polyclonal antibody  at 1 µg/ml in PBS followed by the addition of rVP6 (1 µg/ml in PBS). After washing the unbound rVP6, 10% fecal suspensions , VW samples (diluted 1:5 for IgG detection and 1:2 for IgA detection) or serum (diluted 1:2) were added and the plate was developed with 1:4000 diluted HRP conjugated goat anti-mouse IgG or IgA (both from Sigma-Aldrich) and OPD substrate.°C and added to secretor positive human saliva type A  or type O (for GI-1 VLP binding) coated 96-wells plates. VLPs lacking the serum were used for maximum binding of VLPs to the saliva. The VLPs bound to histo-blood group antigens (HBGAs) present in saliva were detected with NoV antibody positive human serum [Saliva-based blocking assays were used as a surrogate neutralization assay for NoV  and the an serum  and antiin vitro was determined by an ELISA-based RV antigen reduction neutralization assay (NELISA) as described by others [°C the mixtures were overlaid to confluent MA104 cell monolayers in 96-well cell culture plates  following centrifugation for 60 min at 1000 × g. The virus inoculum was replaced with a culture medium containing trypsin (Sigma-Aldrich) at 4 µg/ml and the plates were incubated for 15 h at +37°C. After lysing the cells with a thaw freeze cycle the RV detection in duplicate samples was performed by a Ridascreen® kit  according to the manufacturer’s instructions. A reduction in OD value greater than 60% compared with the positive control wells (trypsin activated RV without the test sample) was considered to indicate neutralization. Neutralizing titers were expressed as the highest sample dilution yielding neutralization.The ability of mucosal and serum antibodies to abolish RV infectivity y others ,43 with y others ) RV stra6 cells/well) for NoV-specific INF-γ production. For detection of RV-specific IFN-γ producing cells, group-wise pooled splenocytes were stimulated with VP6 derived 18-mer peptide previously identified as a VP6-specific CD4+ T cell epitope  [°C and 5% CO2 after which the cells were discarded and the plates were thoroughly washed with PBS. Biotinylated anti-mouse IFN-γ monoclonal antibody  was added and the plates incubated for 2 h at RT. After washing, 1:1000 diluted streptavidin-ALP (Mabtech) was added and the plates were incubated for 1h. The spots were developed with BCIP/NBT substrate (Mabtech) and the formation of color reaction stopped with tap water. The spots were counted by an ImmunoSpot® automatic CTL analyzer  and the results are expressed as mean spot-forming cells (SFC) per 106 splenocytes of duplicate wells.NoV and RV-specific T cell responses were analyzed by quantification of IFN-γ production from splenocytes by ELISPOT  with sli242-259)  at 5 µg/242-259) ) at a VPU-test was used to assess the statistical difference between non-parametric observations of two independent groups. Statistical analyses were done by IBM SPSS Statistics -software  version 19.0 and the statistical significant difference was defined as p ≤ 0.05.A Mann–Whitney The assembly conformations of NoV GII-4 VLPs, GI-3 VLPs and RV rVP6 were verified by transmission electron microscopy (TEM) as described previously . As illuThe optimal amount of antigens to be used in the trivalent vaccine was pre-determined by a dose response study in BALB/c mice immunized with 3, 10 and 30 µg of NoV GII-4 VLPs, GI-3 VLPs or RV rVP6 as single antigens. The dose responses to each antigen were screened by measuring antigen-specific serum IgG antibody titers in ELISA. All three antigens induced robust systemic IgG responses in mice  and the immune responses induced in each group were compared at study week 5. The duration of the immune response induced by the trivalent formulation was followed in another group of mice terminated at study week 24. All antigens induced a robust homologous IgG response  and genogroup I (GI-1) not included in the immunization. GII-4 and GI-3 VLP immunizations induced high levels (mean OD>1.5) of cross-reactive antibodies against VLPs belonging to the same genogroup and significantly lower levels  of antibodies against the VLPs belonging to the other genogroup  and heterologous (non-immunogen-specific) NoV VLPs binding to the saliva HBGAs with mice antiserum . The experiments were repeated several times with consistent results.To detect the functionality of VP6-specific antibodies fecal suspensions, VWs and sera were used to inhibit RV infectivity on assay ,43. Our on assay . Therefoon assay ), homoloNoV and RV-specific IFN-γ producing cells were quantified from mice splenocytes by an ELISPOT assay  stimulates strong systemic cross-reactive antibody responses to both viruses with inter NoV genogroup neutralizing ability; 2) induces mucosal antibodies able to inhibit RVs infectivity; and 3) activates the cellular arm of the immune responses to both viruses. Importantly, all the immune responses induced by the trivalent vaccine were long-lasting and no mutual interference and/or inhibition of the vaccine components in the formulation was observed. The results obtained here are encouraging and support the development of a non-live subunit combination vaccine against NoV and RV for humans.