Approximately 50% of patients with hepatitis C virus (HCV) genotype 1 treated with peginterferon alfa-2a/ribavirin discontinue treatment early or experience a suboptimal response despite 48 weeks of therapy. The objective of this analysis was to develop a model to identify nonrapid virologic response (non-RVR) patients who may be candidates for intensified therapy that would increase treatment response. The retrospective analysis included non-RVR patients from four trials of 48-week peginterferon alfa-2a/ribavirin treatment. Patients were grouped into those who cleared virus between weeks 5 and 12  or between weeks 13 and 24 (slow responders). A model was developed to predict relapse at the end of follow-up (week 72). An optimal model was evaluated and compared with current practice by using receiver operating characteristic curves, sensitivity and specificity. In total, 539 non-RVR patients were eligible for analysis of which 72% experienced cEVR and 28% were slow responders. Variables associated with relapse included age, ethnicity, baseline HCV RNA and interval of time to HCV RNA undetectable. The optimal model was most accurate at predicting patients at risk for relapse. The practice of considering treatment intensification (e.g. extending treatment duration) in all slow responders was less accurate but likely most practical. A week 4 HCV <2-log reduction was the earliest but least accurate marker. We developed a model that could identify non-RVR patients at high risk for relapse after 48 weeks of peginterferon alfa-2a plus ribavirin and who may benefit from intensified therapy to reduce this risk of relapse. Hepatitis C virus (HCV) accounts for approximately 40% of liver-related mortality in the United States . EradicaThe ability to predict more accurately the response to treatment is important. In addition to allowing the customization of therapy, predicting response may help reduce the significant adverse events and high cost associated with this therapy. The probability of achieving SVR can be estimated by considering several baseline patient and viral characteristics of which viral genotype genotype 2 or 3) and pretreatment viral load (<800 000 IU/mL) have emerged from multivariate analyses of large pivotal trials as two major favourable predictors of SVR  or 3 and.More recently, early viral kinetics have been shown to be more highly predictive of SVR than baseline factors . ApproxiAnalysis included treatment-naϊve adults infected with HCV genotype 1 from four clinical trials ,3,13,14.Besides the criteria mentioned above, patients were eligible for this analysis if they were randomized to treatment for 48 weeks with peginterferon alfa-2a 180 μg/week plus ribavirin 1000 or 1200 mg/day, initially became HCV RNA undetectable between weeks 5 and 24, were HCV RNA undetectable at week 48 and had a HCV RNA measurement at week 72. All patients who met the above criteria completed 48 weeks of treatment except for two patients who discontinued early because of an adverse event or illness. These two patients were excluded from the analysis.Depending on the virologic response at week 72, this analysis included non-RVR patients who either achieved SVR or had a confirmed relapse. Patients were then classified as those with cEVR (HCV RNA undetectable at week 12) or those with slow response (HCV RNA undetectable for the first time between weeks 13 and 24).post hoc analysis of data collected during four randomized, multinational, phase III/IV studies where the primary efficacy end-point was SVR, defined as undetectable serum HCV RNA at the end of a 24-week follow-up phase. Undetectable HCV RNA was defined as <100 copies/mL (Cobas® Amplicor® PCR HCV Test) or <50 IU/mL (Amplicor® HCV Monitor Test) in the three earlier studies [This study involved the  studies ,13, and  studies .The outcome variable for this analysis was virologic relapse that was defined as HCV RNA detectable at week 72 following HCV RNA being undetectable at EOT.vs 24-week undetectable), HCV RNA reduction at weeks 4, 12 and 24, and average HCV RNA reduction at nadir. Average HCV reduction at nadir (log10 per month) was calculated as follows: for cEVR, the average HCV reduction at nadir = (log10 HCVbaseline − log10 HCVweek12)/3 and for 24-week undetectable, the average HCV reduction at nadir = (log10 HCVbaseline − log10 HCVweek24)/6. Viral load measurements in copies/mL were converted to IU/mL by using the conversion factor 2.7 (2.7 copies/mL = 1 IU/mL).Candidate predictors that were potentially associated with relapse and were therefore incorporated into the analysis included: patient demographic and clinical characteristics at baseline, time to first HCV RNA undetectable (i.e. cEVR P < 0.20 for elimination. The models obtained after this backward selection were further refined for simplicity and robustness. Percentage dose reduction (as a continuous variable) because of safety reasons was incorporated into the optimal model using a backward selection procedure that eliminated the insignificant (P > 0.05) dose reduction variables one at a time.Logistic regression analysis was used to develop a model to predict relapse at week 72. Descriptive statistics (e.g. percentages and means) of the candidate predictors were reviewed and their relationship with relapse was evaluated using simple logistic regression one at a time. Models were selected starting from a full model which included all candidate predictors and their meaningful interactions . A backwThe selected models were evaluated using receiver operating characteristic (ROC) curves, sensitivity (% of patients correctly predicted as relapse) and specificity (% of patients correctly predicted as SVR). To demonstrate further the improvement in prediction, selected models were compared with the practice of intensifying treatment for all slow responders and leaving cEVR patients without treatment beyond 48 weeks.n = 388) became HCV RNA undetectable during weeks 5 and 12 (cEVR), and 28.0% (n = 151) became HCV RNA undetectable during weeks 13 and 24 (slow responders). Demographic and clinical characteristics of the total population are presented in In total, 539 non-RVR patients who became HCV RNA undetectable before week 24 were eligible for inclusion in this analysis, of which 72.0% (10 per month).Simple logistic regression analysis was performed on each of the candidate predictors as an initial evaluation of a relationship with relapse. Results  and 4 suUsing a backward selection procedure starting with the full model, several less complex multivariable logistic models containing fewer variables were developed to predict relapse. The variables included in the optimal model and the parameter estimates are presented in A ROC curve derived from the optimal model was far from the diagonal straight line indicating that the optimal model was valuable in predicting relapse . It is kvs 47%, respectively, and a specificity of 82%vs 74%, respectively  that are known to influence HCV therapeutic end-points, we developed a model that improved the accuracy of identifying non-RVR patients who responded by week 24 yet were unable to achieve SVR in this retrospective cohort. The model could identify a subset of patients at high risk for relapse and who could then be considered for intensified therapy. Thus, the use of this model could potentially lead to an increase in SVR rates as well as prevent unnecessary intervention in those patients who would not relapse even without intensified therapy.n = 230) or 72 (n = 225) weeks. However, a subanalysis of this cohort revealed that those patients with detectable HCV RNA at week 12 experienced higher SVR rates with 72 weeks of therapy compared with those patients who received only 48 weeks of therapy  [et al. [n = 326) and without RVR were randomized to receive either 48 or 72 weeks of peginterferon alfa-2a 180 μg/week plus ribavirin 800 mg/day. Although EOT responses were similar, non-RVR patients with HCV genotype 1 who received 72 weeks of therapy were much less likely to relapse than patients with 48 weeks of therapy , resulting in a significant difference in SVR rates between the two groups  [Several strategies that intensify therapy have been explored to improve therapeutic outcomes. Extending treatment duration beyond 48 weeks is one such strategy that has been recently investigated. However, if applied to the majority of patients, this regimen will potentially place many patients at risk for unnecessary treatment-related adverse events. Not all investigators agree that extending treatment duration is an effective strategy for the majority of patients. Berg and colleagues  showed t = 0.04) . A similvs 45% for the 48-week arm; P-value not significant), the rate of SVR was superior in patients treated for 72 weeks  [Accurately identifying candidates for intensified therapy is crucial to minimize both cost and morbidity. We developed a multiple logistic regression model that could identify non-RVR patients likely to relapse during the 24-week follow-up period after treatment is completed. The optimal model provided acceptable sensitivity and specificity to identify patients who may relapse and therefore may benefit from intensified therapy. Age, ethnicity, time to first HCV RNA undetectable, baseline ALT level, baseline HCV RNA level, advanced fibrosis and the degree of HCV RNA reduction at weeks 4 and 12 contributed to the risk for relapse and were included in the model. However, as computation is slightly laborious, the optimal model was compared with the more simplistic but clinically practical approach of intensifying therapy in all patients who are slow to respond to combination therapy and become HCV RNA undetectable between weeks 13 and 24. This approach has also been prospectively evaluated. Pearlman and colleagues  randomiz= 0.026) . More re= 0.026) –17, incl= 0.026) . In our IL28B) gene that strongly predict the response to current HCV therapy [IL28B genotyping in identifying patients likely to respond to HCV therapy is expected to improve patient care in the near future. Multiple logistic analyses that included baseline (pretreatment) clinical predictors of viral response showed that several IL28B genetic polymorphisms were independent predictors of response [IL28B rs12979860 CC genotype was the strongest pretreatment factor associated with SVR and had a higher adjusted odds ratio than HCV genotype, viral load, age, sex, race, fibrosis or previous treatment status [IL-28B for SVR is limited to its use as a baseline pretreatment characteristic only. It is well known that the on-treatment virologic response to HCV therapy is a stronger predictor for SVR than baseline characteristics [et al. also reported that although this genotype did not contribute additional information to patients with RVR, the CC genotype added significant predictive value in patients with non-RVR; however, once the week-12 virologic response was determined, the predictive utility of this genotype was very weak [IL28B genotype on the proposed models in our study; however, because DNA samples were not collected during the trials included in this analysis, we cannot comment on what the effect of adding IL28B polymorphisms to our models would have had on their specificity or predictability.Recent studies have identified genetic variations in or near the interleukin 28B ( therapy –22. The response –22. Subst status ,24. Thist status . These seristics . For exaeristics ,6,9. In eristics . ThompsoThere are advantages to the early identification of a patient in need of alternative therapeutic strategies. Combination therapy is associated with significant adverse events that affect family planning, employment and health-related quality of life. Although RVR is highly correlated with SVR, early viral kinetics have not been used to predict suboptimal response. The week-4 model of intensifying treatment in all patients with a <2 log decrease in HCV RNA at week 4 and who were HCV RNA undetectable by week 24 was the earliest marker of candidates for intensified therapy, but was also the least accurate. However, this time point may be advantageous if considering alternative therapeutic strategies such as high-dose ribavirin or the addition of a third agent. Although the proposed model was developed using data from patients treated with peginterferon alfa-2a plus ribavirin, given the fact that 20–30% of patients relapse after treatment with either of the currently approved pegylated interferons, it is likely that this model is applicable to all patients with HCV genotype 1 undergoing standard HCV therapy .In conclusion, two steps are necessary to achieve the goal of increasing the therapeutic efficacy in patients who may relapse on standard HCV therapy. First, the patient would have to be accurately differentiated from those patients who are likely to achieve SVR on standard therapy alone. Second, the identified patient would then have to receive an intensified therapy that has been proven in prospective clinical trials to increase therapeutic efficacy. The multiple logistic regression model developed in this study accomplishes the first step of identifying non-RVR patients who may relapse during the 24-week follow-up period after treatment with standard of care of combination peginterferon alfa plus ribavirin. However, the external validity of this model can only be achieved by evaluating the sensitivity and specificity after applying it to a separate database of patients who received similar treatments. This process will ensure the robustness of the model and that this model can be generalized to include other interferon-based therapies. The model predicts those patients at risk for suboptimal response at a time when the on-treatment regimen may still be modified by strategies to intensify therapy and improve treatment outcomes. Once the applicability of the model across all interferon-based therapies is established, these results may guide individualization of treatment decisions in clinical practice, and ultimately, the model may be used to develop a nomogram that will assist physicians in determining whether to treat a patient with intensified therapy.


Global prevalence of Hepatitis C Virus (HCV) infection corresponds to about 130 million HCV positive patients worldwide. The only drug that effectively reduces viral load is interferon-α (IFN-α) and currently combination of IFN and ribavirin is the choice for treatment.The present study is aimed to resolve the genotypes based on core gene that might affect the response to interferon therapy. Furthermore an attempt was made to propose a powerful therapeutic approach by designing the siRNA from sequences of the same patients who remain resistant to IFN in this study.To achieve the objectives, a sequence analysis was performed in five HCV ELISA positive subjects who have completed IFN treatment. Neighbor Joining (NJ) method was used to study the evolutionary relationship. Atomic models were predicted using online software PROCHECK and i- TASSER.Two new genotypes were reported for the first time namely 4a from suburban region of Rawalpindi and 6e from all over the Pakistan. According to Ramachandran plot, satisfactory atomic model was considered useful for further studies, i.e. to calculate HCV genotypes conservation at structural level, to find out critical binding sites for drug designing, or to silence those binding sites by using appropriate siRNA. Single siRNA can be used to inhibit HCV RNA synthesis against genotype 3 and 4, as the predicted siRNA were originated from the same domain in studied HCV core region in both genotypes.We can conclude that any change or mutation in core region might be the cause of HCV strains to resist against IFN therapy. Therefore, further understanding of the complex mechanism involved in disrupting viral response to therapy would facilitate the development of more effective therapeutic regimens. Additionally, a single designed siRNA can be used as an alternative for current therapy against more than one resistant HCV genotypes. Hepatitis C virus (HCV) is endemic worldwide infection and its distribution varies geographically. Prevalence estimate is much higher among developing countries than in developed countries . PakistaPhylogenetic analyses identified six major lineages, namely genotype 1-6. These groups were further subdivided into several subtypes. The creation of quasispecies is possible due to high rate of mutation in hepatitis virus strains, even within a single infected individual. On the basis of genetic similarities, numerous viral strains have been categorized into groups, types, and subtypes. Genotype 1 is the most common lineage in North and South America as well as in Europe . HoweverPractice of siRNA is more valuable as it binds directly to specific mRNA results in exclusive block of transcription potentially bearing a powerful molecular therapeutic approach rather than current therapy of highly liable to RNAi-induced suppression, as the inhibition of HCV RNA levels by targeting different genes using RNAi has been reported . HCV CorIn the present study the genotypes of five HCV positive but unresponsive to IFN therapy were resolved. Further, an attempt was performed to design an anti-HCV siRNA in studied samples to be employed against any HCV genotype.HCV Enzyme linked immune sorbent assay (ELISA) positive individuals who have completed IFN treatment were randomly selected from Rawalpindi General Hospital. Blood samples were collected from patients after taking informed written consent.Qualitative detection of HCV RNA was performed using Reverse transcriptase (RT) PCR. Briefly, 150 μl of patients’ sera samples were used to isolate the RNA by a commercially available kit (NucleoSpin RNA Virus by Machereynagel) according to the manufacturer’s instructions. Complimentary DNA (cDNA) of partial core region HCV was synthesized using 100 units of Moloney murine leukemia virus (MMLV) reverse transcriptase enzyme  with 10 μM of outer antisense primer. Two PCR amplification cycles were performed (first cycle PCR and nested PCR) with five units of Taq DNA polymerase enzyme  in a volume of 25 μl reaction mixture. External PCR conditions performed in a thermal cycler were as follows: an initial denaturation step at 95°C for 3 minutes followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, and finally extended at 72°C for 3 minutes. Internal PCR conditions were the same except for a different set of inner primers used with annealing temperature for 5 minutes in cycle 1 of PCR amplification. Products of nested PCR were directly sequenced on Beckman Coulter CEQ 8000 sequencer after purification by PCR Product Purification Kit from Genomed.Five obtained sequences were aligned by ClustalW and similarity of sequences with which already reported in database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was found by Nucleotide Blast (nBlast). . After aProtein structure and function was predicted using i-TASSER server after translating the nucleotide sequence into amino acid. 3D models were built based on multiple-threading alignments and i-TASSER assembly simulations; function insights were then derived by matching the predicted models with protein function database 24]..24].3D structural models built using i-TASSER were evaluated on PROCHECK. It checks the stereochemical quality of a protein structure that produces a number of plots in PostScript format analyzing its overall and residue-by-residue geometry. Pdb files of 3D models were uploaded and Ramachandran plot was used to check the existence of five models predicted by i-TASSER for each sequence.Antiviral siRNA prediction was made for HCV core region. SiRNA sequences were selected based on their degree of conservation, defined as the proportion of viral sequences that were targeted with complete matches by corresponding siRNA. SiDirect was employed to predict siRNA as a highly effective, target specific siRNA online design tool. Five sequenced HCV samples were pasted in FASTA format and the program was run by implementing Ui-Tei algorithms, the algorithms that are on the back hand of siDirect . The outPartial sequencing of core region of five HCV positive samples resistant to IFN therapy  revealed different genotypes mentioned in Aligned sequences using multiple alignment software Clustal W were further subjected to construct tree using the software MEGA 5 to find out the relationship between the sequences, novel genotypes, subtypes, and variants. Unrooted NJ tree constructed for studied samples with reference sequences  from Los Alamos mounted from two main clades that show the presence of separate genetic lineages. Clade 1 showed all samples of the present study while clade 2 exhibited irregular pattern of evolution among studied samples representing different geographical regions worldwide however separated in different clusters . Active Models for the studied sequences were first built by the use of i-TASSER which generates up to five full-length atomic models (ranked based on cluster density) of the studied samples with resolved genotypes. Ramachandran plots were built using PROCHECK to evaluate these models for 3D structural analysis . The nexModels predicted by i-TASSER with highest C-score as well as satisfactory plot statistics (< 75 %) can be further used for 3D models analysis which could help us to predict the cleavage sites or recognition of phosphorylation sites.Tajima’s test of neutrality collates the number of discriminating sites per site with the nucleotide assortment. A site is considered segregating if in a comparison of sequences, there are two or more nucleotides at that site. The Tajima test was calculated using MEGA 5. All gaps were eliminated from the data group; m = number of sites, S = number of segregation sites, Ps = S/m, θ = ps/a1, and л= nucleotide diversity. D is the statistical test result.Highly effective siRNA sequences were selected by using novel guidelines that were established through an extensive study of the relationship between siRNA sequences and RNAi activity by online available software siDirect. The siRNA was predicted against five HCV samples that were resolved in the present study . The preGenotype is one of the strongest predictive aspects of sustained virological response . In the The NJ tree was constructed for the studied samples with reference sequences from the (LANL) mounted by two main clades showed the presence of separate genetic lineage. The Dendrogram was developed to find an association between studied samples and reference accessions from other countries; overall, it was revealed that the studied HCV samples of the present study exhibited long branch lengths, indicating an ancient history of their evolution and their genetically stable genome composition; this might be attributed to the suppressed or compromised immune pressure of the host. Previous reports supported the evidence that cases where immune response is compromised, there are a less chance of viral clearance 44]. Sec. Sec44].Test values indicated that the high mutation rate of the HCV might be one of the points of determinant action of the natural selection and thereby cooperated in inducing the divergence of viral species. At the beginning of HCV infection, there is a reduction in the viral population . DespiteProtein models were predicted using i-TASSER of the studied sequences. These 3D structures would be helpful in predicting the cleavage sites or recognition of phosphorylation sites. After translation is completed, during HCV replication, HCV polyprotein gets cleaved into at least ten distinct products. The order in which cleavages occur from N-terminus to C-terminus is - C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B . These pThe siRNA was predicted against five HCV samples that were resolved in the present study. Different domains of core perform different functions like siRNA predicted against all the genotypes 3a, 4a and 3b) positioned at N-terminal from 13-35 nucleotides contains RNA and DNA binding domain [3][4][5]a and 3b


Knowledge on long-term preservation of micro-organisms is limited and research in the field is scarce despite its importance for microbial biodiversity and biotechnological innovation. Preservation of fastidious organisms such as methane-oxidizing bacteria (MOB) has proven difficult. Most MOB do not survive lyophilization and only some can be cryopreserved successfully for short periods. A large-scale study was designed for a diverse set of MOB applying fifteen cryopreservation or lyophilization conditions. After three, six and twelve months of preservation, the viability (via live-dead flow cytometry) and culturability  of the cells were assessed. All strains could be cryopreserved without a significant loss in culturability using 1% trehalose in 10-fold diluted TSB (TT) as preservation medium and 5% DMSO as cryoprotectant. Several other cryopreservation and lyophilization conditions, all of which involved the use of TT medium, also allowed successful preservation but showed a considerable loss in culturability. We demonstrate here that most of these non-culturables survived preservation according to viability assessment indicating that preservation induces a viable but non-culturable (VBNC) state in a significant fraction of cells. Since this state is reversible, these findings have major implications shifting the emphasis from survival to revival of cells in a preservation protocol. We showed that MOB cells could be significantly resuscitated from the VBNC state using the TT preservation medium. Long-term preservation of micro-organisms is an often neglected but very important aspect of both applied and environmental microbiology Upon resuscitation (after cryopreservation) or rehydration (after lyophilization), a preservation method is considered successful when strains are culturable  after preservation, preferably in high numbers (quantitatively), as evaluated by counting either most probable numbers (MPN) or colony forming units (CFU) The aim of the present study was to evaluate the success of long-term preservation of a diverse set of MOB by applying 15 preservation conditions using both lyophilization and cryopreservation. Viability was assessed through live/dead flow cytometry while culturability was evaluated through MPN analysis and plating. Since both viability and culturability were assessed quantitatively, it was explored whether non-culturable cells were either dead or still viable and whether the VBNC fraction could be reduced by resuscitation efforts. The obtained results showed that all MOB could be successfully preserved by cryopreservation and lyophilization, and demonstrated that a VBNC state induced by preservation could be significantly reduced using a specifically adapted preservation medium.Alphaproteobacteria (Type II MOB) and the Gammaproteobacteria (Type Ia/b MOB) www.dsmz.de and www.ncimb.com) and their main characteristics are listed in T was cultivated in DSM1180 medium (pH 9.0) (www.dsmz.de). Cultures were grown either in broth with 20% (v/v) CH4 added to the headspace or on solid medium in gas-tight jars  under a CH4:air (1∶1) atmosphere.Most of the aerobic methane-oxidizing diversity obtained so far is phylogenetically positioned within the Prior to preservation, the MOB strains were grown in broth at optimal temperature  to early−2 up to 10−9) was prepared in triplicate in 96-well microtiter plates (200 µL/well) and incubated in dNMS or DSM1180 medium at optimal temperature (4:air (1∶1) atmosphere. After one and two weeks of incubation, growth was monitored by measuring the optical density at 600 nm  and most probable numbers were calculated using MPN-Tables −1, 10−3 and 10−5 in duplicate (50 µL/plate) on dNMS or DSM1180 medium and incubated as mentioned higher. The amount of colony forming units per volume (CFU/mL) was estimated after one and two weeks of incubation. For logistic reasons, culturability was quantified by MPN for the eight strains grown at 28°C, and by plate counting for the remaining two strains.To enumerate the culturable MOB at early stationary phase, either most probable number (MPN) or plate counts were performed. For MPN counts, a dilution series (10perature  in gas-t4–106 cells/mL) for FCM measurements and 10 µL mL−1 live/dead stain, 5 mM Na2EDTA and 10 µL mL−1 Cytocount™ beads at 1,064 particles µL−1  were added. The live/dead stain was composed of 30 mM propidium iodide (Invitrogen Switzerland) mixed with SYBR®Green I (10−2 dilution in dimethyl sulfoxide (DMSO)) at a ratio of 1∶50. Following staining, samples were mixed thoroughly and incubated in the dark for 15 min at room temperature. Samples were analyzed using a CyAn™ High Performance Flow Cytometer  equipped with a 50-mW sapphire solid-state diode laser, emitting at a fixed wavelength of 488 nm. The optimal settings for each strain were determined as described by −1 (based on bead counts) of the MOB cultures was calculated.MOB were quantified by live/dead flow cytometry (FCM). The cultures were diluted to optimal cell density . The six LPA combinations are listed in ™ vials  were used to add 500 µL sample to 500 µL cryoprotectant (CPA). The eight CPA combinations are listed in ™ beads . After centrifuging  1 mL of each culture grown in carbon-deficient media, the cell pellet was harvested using sterile cotton swabs and beads were inoculated with cell material according to the manufacturer’s instructions. The vials were preserved at −80°C.The ten MOB type strains cultivated as explained above were subjected to long-term preservation by cryopreservation in liquid nitrogen. CryotubeThe lyophilized suspensions were rehydrated after 3, 6 and 12 months. Ampoules were opened and 1 mL fresh medium  was added to rehydrate the cells and the suspensions were transferred to eppendorf tubes. After a rehydration step at room temperature for approximately one hour, the culturable and viable recovery of the cells was evaluated as above.™ beads was evaluated on plates according to the manufacturer’s instructions. Since quantification is not possible using the beads, culturability was only assessed qualitatively for this preservation condition.The cryopreserved suspensions were resuscitated after 3, 6 and 12 months. Vials were thawed at 37°C, and upon thawing, immediately transferred to eppendorf tubes and centrifuged . After supernatant removal, an equal volume of fresh medium  was added to avoid toxic effects of DMSO and methanol at room temperature. After a resuscitation step at room temperature for approximately one hour, the culturable and viable recovery of the cells was evaluated as above. The survival of the cultures on Microbank4 in the atmosphere. In addition, 16S rRNA gene sequence analysis was performed on the cultures, as described previously Prior to preservation, the ten MOB type strains were verified for absence of heterotrophic satellites on trypticase soy agar after 2 weeks of incubation without any additional CHPrior to statistical analysis, recorded measurements were log transformed. Imputation of missing values was performed by implementing the expectation-maximization (EM) maximum likelihood estimation (MLE) algorithm, with the maximum number of iterations set at 1000. Nested analysis of variance (ANOVA) designs with repeated measures were used for evaluating the possible effect of the duration of preservation and the effect of LPA or CPA conditions used on culturability and viability measured by MPN and FCM, respectively. Depending on whether or not the assumption of sphericity was respected, no correction  or a Greenhouse-Geisser or Huynh-Feldt correction  was taken into account. ANOVA implementing Scheffé’s post-hoc test was used to assess which preservation condition scored significantly better than others. The effect of adding TT was assessed using a paired samples T-test. All statistical data analyses were performed using IBM SPSS Statistics 19 .Currently, there is no universal protocol for the long-term preservation of methane-oxidizing bacteria. Therefore, a diverse set of ten type strains of MOB, representing eight genera and the three major MOB types , was tested using 15 preservation conditions . The conT, which was not tested in TT medium  per preservation condition for each of the strains are shown in Bacillus cereus LMG 6923T and Pseudomonas aeruginosa LMG 1242T that do not demonstrate VBNC due to their high recovery of culturability (data not shown). Injured cells mimicking the VBNC state, and thus not truly dormant cells The essence of preserving biomass is the possibility to again grow cells when needed. Therefore, cultivation is the routine evaluation tool of preservation success. Unfortunately, this approach does not allow differentiation between cell death and survival of cells unable to replicate in given cultivation conditions (i.e. VBNC cells). The VBNC state is known to be induced by stress but has thus far never been linked to preservation. Here, we unequivocally demonstrated that recovery of culturability and not viability of biomass after preservation is most problematic: a significant discrepancy between viability and culturability after preservation was observed for ten MOB type strains  in fifteen different preservation conditions analyzed in parallel by live/dead flow cytometry and cultivation, respectively. To overcome possible subjective and difficult interpretation of FCM data . The presented results indicate that by applying appropriate preservation strategies as also discussed thoroughly by Tindall In conclusion, the present study demonstrated that recovery of culturability and not viability is the bottleneck for reviving preserved MOB. Therefore, improved resuscitation procedures should become a key aspect of preservation protocols. It was shown here that the use of a combination of TSB and trehalose in the growth, preservation and resuscitation medium with 5% DMSO as cryoprotective agent significantly reduced the induced viable but non-culturable state. Finally, this was further confirmed by successfully preserving 22 recently isolated MOB Table S1The log drop in viability and culturability and the VBNC fraction for each strain tested with each preservation condition. The VBNC fraction was calculated by subtraction of the culturable drop by the viable drop. Detection limit values were used to calculate drops with counts that were below this limit, such calculations are marked with ≥, indicating that the true drop is potentially even higher. Calculations of drops that resulted in negative values were indicated as 0, since these would implicate an impossible increase after preservation, attributed to variations in the measurements.(DOC)Click here for additional data file.


A fundamental life history question is how individuals should allocate resources to reproduction optimally over time . The reproductive restraint hypothesis predicts that reproductive effort  should peak at prime-age, whilst the terminal investment hypothesis predicts that individuals should continue to invest more resources in reproduction throughout life, owing to an ever-decreasing residual reproductive value. There is evidence supporting both hypotheses in the scientific literature.Rupicapra rupicapra) shot at various times during the rutting period to test these two hypotheses. We assumed that body mass loss in rutting males was strongly related to RE and, using a process-based approach, modelled how male relative mass loss rates varied with age. For different regions of our study area, we provide evidence consistent with different hypotheses for reproductive allocation. In sites where RE declined in older age, this appears to be strongly linked to declining body condition in old males. In this species, terminal investment may only occur in areas with lower rates of body mass senescence.We used an uncommonly large, 38 year dataset on Alpine chamois ; both could explain a decline in RE in old age and may be hard to disentangle in empirical data. We discuss a number of environmental and anthropogenic factors which could influence reproductive life histories, underlining that life history patterns should not be generalised across different populations. In most stable populations, reproductive life histories should be optimised to maximise lifetime reproductive success The classical view of reproductive allocation in iteroparous species is that reproductive effort  should increase throughout life following maturity, due to a decreasing residual reproductive value Evidence from a number of species suggests that RE peaks at prime-age (here defined as the age of peak body condition) and subsequently decreases Ungulate species provide good model systems with which to study the allocation of RE over time. Males and females generally have strongly contrasting life histories, investing in reproduction in different ways and over different periods of the reproductive cycle Cervus elaphus) before declining; this is probably explained by a reduced ability of older males to hold a harem Bison bison) Oreamnos americanus) Alces alces), although the authors noted that effort tended to stabilise or decrease in the oldest individuals (which were sparse in their dataset). Unfortunately, several of these studies were based on small sample sizes, particularly of individuals of prime-age and older, a common problem in life history studies Reproductive allocation by males has been studied in several ungulates, using both transversal (cross-cohort) and longitudinal approaches. Using a transversal hunting dataset, Yoccoz et al. Rupicapra rupicapra). During the rut, males allocate most of their non-rutting time to rest rather than foraging; they are thus capital breeders Here, we analyse the relationship between male age and RE using a large transversal dataset of Alpine chamois . The elevation of the study area ranges from 52 to 3,558 m above sea level, with a mean altitude of 1,586 m. It is forested up to the tree-line at about 2,000 m, above which habitat consists of Alpine meadows and open rock faces. The study area consists of six chamois hunting districts  culled over 38 consecutive hunting seasons between 1973 and 2010. Ages were estimated from counts of horn growth annuli d, is only a crude index but generally correlates well with population density values estimated from censuses performed since 1992  is t (t≥300) isWe sought evidence for age-dependence in the relative rate of male mass loss during the rutting period. We recognise that annual population density is a potentially important factor affecting the rate of mass loss and may vary across years at a given site nce 1992 . We deveOur objective is to estimate We assumed that age-dependence for both the initial mass and mass loss functions could be characterised using cubic splines. Cubic splines were used to avoid numerical instabilities that are often characteristic of higher order polynomials [e.g. 38]. We assumed that the shape of these splines was fixed across years for any site (i.e. age-dependence was fixed) but that their relative magnitude could vary among years to reflect annual environmental variation. Specifically, we used three cubic splines, each spanning 4 years of age, to describe complete age-dependency from 1 to 13 years of age. These three splines correspond broadly to pre-prime age (1–4 years), prime-age (5–9 years) and post-prime age (10–13 years). There were too few individuals older than 13 within each site to permit reliable predictions for these individuals. Using fewer than three splines resulted in a poor fit to our data, whereas using more than three splines did not improve the fit for either function. Initial mass, and mass loss rate, were described byandf(a) and g(a) describe the two three-piece, cubic-spline functions and satisfy f(1) = g(1)  = 1. Thus, the scaling parameter 0α represents the inverse of the initial mass of the youngest age modelled (yearlings). Similarly, 0β describes the inverse of the rate of mass loss of yearlings. To identify long-term trends and density-dependence, models were constructed that allowed 0α and 0β to be either: constant, linearly dependent on d, linearly dependent on y, or quadratic in y. Incorporating yearly trends and density-dependence in life history traits is plausible for long-term studies of mammalian life histories. In total, we fitted 16 models to both male and female mass time-series data for each of the three sites . Second, in order to remove overly complex models, we disregarded those that had a higher AIC value than any simpler nested model.When fitting the models we assumed that variation about the predicted mean (eqn. 1) was normally-distributed. Models most consistent with the data were selected using Akaike's Information Criterion AIC; . To avoiIgnoring site effects substantially reduced the fit of our models, justifying fitting separate models for each site  & S4. Fo0,α varied with year  differed between sites Click here for additional data file.Figure S2Variation in relative day shot with age in males and females. Relative day shot was calculated for each individual by shooting day minus median day of shot animals in the same year and site. Thick lines represent median values, boxes display inter-quartile ranges (IQRs) and the extents of the vertical dashed lines show maximum and minimum values. Outliers represent values more than 1.5 of the IQR higher or lower than the mean and, in such cases, the extents of dashed lines represent maximum and minimum values within 1.5 of the IQR. Non-overlapping notches on boxes provides strong evidence that the medians of these age classes differ. Note that, if artificial selection  was occurring, we would expect to see a U-shaped curve in these plots ; this is not the case.(TIF)Click here for additional data file.Figure S3The relationship between d, our population density estimate, and population density estimated from censuses. Linear regressions plotted. Fitted line equations and R2 values shown.(TIF)Click here for additional data file.Figure S4Plots of model fit for males. The relationship between observed body mass and predicted body mass by best model, in each site. Linear regressions plotted (red lines). Fitted line equations and R2 values shown. Dotted lines show 1∶1 relationship.(TIF)Click here for additional data file.Figure S5Change in predicted yearling male mass over time. Variation between predicted pre-rut body mass of yearling males (on day 300) with year in Presanella (red line), Adamello (black line) and Brenta (blue line). Line equations shown. There is a clear trend of decreasing body mass over the study period.(TIF)Click here for additional data file.Table S1Sex and age-specific chamois hunting quotas in Trento province.(DOC)Click here for additional data file.Table S2Male and females data summary.(DOC)Click here for additional data file.Table S3Model selection results from cubic spline model fitted to male chamois body mass data.(DOC)Click here for additional data file.Table S4Model selection results from cubic spline model fitted to female chamois body mass data.(DOC)Click here for additional data file.


Chironex fleckeri  stings can cause acute cardiovascular collapse and death. We developed methods to recover venom with high specific activity, and evaluated the effects of both total venom and constituent porins at doses equivalent to lethal envenomation. Marked potassium release occurred within 5 min and hemolysis within 20 min in human red blood cells (RBC) exposed to venom or purified venom porin. Electron microscopy revealed abundant ∼12-nm transmembrane pores in RBC exposed to purified venom porins. C57BL/6 mice injected with venom showed rapid decline in ejection fraction with progression to electromechanical dissociation and electrocardiographic findings consistent with acute hyperkalemia. Recognizing that porin assembly can be inhibited by zinc, we found that zinc gluconate inhibited potassium efflux from RBC exposed to total venom or purified porin, and prolonged survival time in mice following venom injection. These findings suggest that hyperkalemia is the critical event following Chironex fleckeri envenomation and that rapid administration of zinc could be life saving in human sting victims. Chironex fleckeri), which displays up to 60 two-meter long, ribbon-like tentacles and inhabits coastal mangroves over a broad and potentially expanding geographic range, spanning 40° latitude from Australia to Vietnam Chironex fleckeri envenomations occur each year, typically from November to May, in North Queensland, Australia Chironex, have been reported recently as far north as Phuket, Thailand The class Cubozoa (phylum Cnidaria) comprises two families (Carybdeidae and Chirodropidae) Alatina moseri  Chironex fleckeri venom (MW 43 and 45 kDa) Envenomation results in painful violaceous skin lesions, and occasionally in death attributed to cardiovascular collapse C. fleckeri venom, calcium channel blockade has been proposed as treatment for such stings. However, pharmacological calcium channel blockers do not prevent calcium influx Currently available treatment options for cubozoan stings are suboptimal. The Commonwealth Serum Laboratory antivenom  is of limited value Chironex are sparse, but available information suggests that a hypertensive phase is followed by hypotension and cardiovascular collapse Detailed clinical data on individuals stung by All experimental protocols using mice were approved by the Institutional Animal Care and Use Committee of the University of Hawaii (Protocols 03-011-4 to 03-011-8), in accordance with Department of Health and Human Services PHS Policy and USDA Animal Welfare Act guidelines. The University of Hawaii has an Animal Welfare Assurance number on file (A3423-01) with the Office of Laboratory Animal Welfare. Human blood used in this study was donated by healthy adult volunteers after providing written informed consent, in accordance with the University of Hawaii Committee on Human Studies policies and specifically approved in protocol CHS # 12561.Chironex fleckeri tentacles  and tentacles of fresh Alatina moseri  by gentle rotation at 4°C in 1 M citrate 1∶4 (v:v) until approximately 90% cnidae were recovered. Sieved (0.5-mm mesh) cnidae solutions were centrifuged at 400 g for 20 min. Undischarged cnidae pellets were resuspended in chilled 1 M citrate at 1∶20 (v:v) and washed twice at 250 g for 20 min, then gently diluted 1∶0.5 (v:v) with ice-cold deionized water to a slurry and transferred to a pre-chilled French Press 20 K pressure cell (SLM-AMINCO Cat# FA078) and subjected to 12,000 psi for 10–15 min. The lysate  was expelled at 30 drops/min and recycled 2–4 times to achieve >90% cnidae rupture, then centrifuged at 12,000 g at 4°C for 5 min. The viscous supernatant (venom) was snap frozen in liquid nitrogen and stored at −80°C. Protein concentrations were determined using a Bradford protein assay (Bio-Rad Protein Assay). Size-exclusion chromatography was performed using a BioSilect 125-5 column (BioRad 125-0060 with BioRad 125-0072) equilibrated with sodium phosphate buffer  at a rate of 0.5 mL/min using an AKTA Purifier high-pressure liquid chromatography (HPLC) system (GE Biosciences).Intact cnidae were recovered from beachside excised 50 unit was defined as that amount of protein required to lyse 50% of RBC in 1 mL of a 1% RBC solution at 37°C in 1 hr. An HU50 unit typically represented about 10 ng total venom protein for A. moseri and 150 ng for C.fleckeri.Hemolytic activity assays were performed in 96-well v-bottom microtiter plates by adding washed 2% red blood cells (RBC), prepared from blood of healthy human donors, to serial two-fold dilutions of total venom and incubating at 37°C for 1 hr, or in whole blood time series experiments with the removal of 0.5 mL aliquots at each specific time point, then immediately centrifuged at 1,000 g for 4 min at 4°C. Supernatants were transferred to fresh flat bottomed plates for 405 nm measurements using a BioRad Ultramark microplate reader . Hypotonic lysis with water served as a positive control and unexposed 2% RBC supernatant as a negative control. Absolute hemoglobin concentrations were determined by converting absorbance at 405 nm using Beer’s law. An HUet al. et al. et al. et al. Alatina moseri tentacles were used. Briefly, for the Winkel preparation, fresh tentacles were washed with PBS, scraped with forceps to yield a solution of nematocysts that was centrifuged; the resultant pellet was resuspended and ground with siliconized glass mortar and pestle, and then cleared of debris by centrifugation Previously published venom preparation methods were followed to compare recovery, toxicity and specific activity of the venom obtained with our new method. Specifically, the methods described by Winkel Potassium concentrations in plasma, whole blood and 2% RBC suspensions were determined in triplicate using a double-junction potassium ion-specific electrode  and 4-channel Ion Analyser Software .50 HPLC-purified Chironex fleckeri or Alatina moseri hemolysin fraction(s) Human RBCs, washed three times in PBS, to a final dilution of 10% (v/v) were added to an equal volume of 2,000 HUChironex fleckeri antivenom effects were examined in venom-exposed RBC and in lethally dosed mice. Package instructions list the recommended dose for humans to be 20,000 U at a dilution of 1∶10. For an adult with a blood volume of 5 L and hematocrit of 40, the final circulating concentration of antivenom recommended would be approximately 0.1 U/mL of blood volume/1% of RBC. Doses above and below this CSL 0.1 U/mL/% were used in this study.The CSL 50 units per blood volume per percent RBC (U/mL/%) based on individual animal body mass to calculate blood volume and published hematocrit value of 44. Control mice were injected with saline only; zinc control mice were injected with a specific calculated volume (approximately 100 µL) of 100 mM zinc gluconate to achieve the targeted circulating concentration, 5 mM; total venom mice were injected with concentrated or saline diluted total venom to achieve the targeted circulating concentration (ranging from 0.5 to 400 U/mL/%); pre-zinc treatment + total venom mice were injected with a volume, as described above, of 100 mM zinc gluconate followed by total venom 60 sec later; total venom + zinc post treatment mice were injected with total venom followed by a volume, as described above, of 100 mM zinc gluconate 60 sec later; and total venom + CSL antivenom post treatment mice were injected with total venom followed by 100 µL 1∶10 diluted in saline (150 mM NaCl) CSL antivenom  for a final dose of 0.26 U antivenom/mL/% 60 sec later. High-resolution M-mode scans and physiological data  were recorded simultaneously. Within 60 sec of death, as determined by loss of ECG activity and respiration, or after CO2-mediated euthanasia, blood was obtained from the left ventricle and plasma was stored at −80°C for subsequent testing. For survival studies without ECHO or ECG, C57BL/6 mice were likewise anesthetized and treated as described.C57BL/6 male mice (22–28 g), lightly anaesthetized with isoflurane (3%), were placed supine on a heated platform with paws secured to built-in ECG electrodes. Body temperature was maintained at 37°C, and isoflurane (1%) and oxygen were maintained via a nose cone. Transthoracic ECHO was performed with a 30-MHz transducer using a Vevo 2100 ultrasound machine . Left ventricular M-mode images were obtained from parasternal short-axis views at the level of the papillary muscle. Saline (150 mM NaCl), total venom or purified porin (at dose designated concentrations with volumes ranging from 15 to 100 µL) was infused via a tail vein catheter (SAI Infusion Technologies) at 200 µL/min, followed by a saline flush. Injected volumes were calculated to achieve specific HUSurvival data were analyzed using GraphPad Prism 5.00 . Differences between groups were determined using the χ2 test (Fisher’s exact test was used instead of χ2 when only two groups were considered) and Kruskal-Wallis statistic  and p<0.05 was considered significant. Survival curves were analyzed according to the Kaplan-Meier method, and for differences between curves the p value was calculated by the log-rank test.Alatina moseri collected locally. The histogram plots summarize the comparative yields in terms of nematocysts (Nem) recovered per animal (50) recovered per microgram of protein . At a dose of 25 U/mL/%, 90% mortality was observed within 23 min, with a mean survival time of 19 min. Injection of a single bolus of zinc gluconate prior to this dose of venom markedly prolonged survival (P = 0.0006) to a mean survival time of 48 min , equivalent to a lethal human sting with 2 meters of Chironex fleckeri tentacle, total venom injection resulted in acute ventricular decompensation associated with conduction anomalies. Within 90 sec, contractility was markedly impaired and subsequently deteriorated further. The ECG showed progressive slowing of sinus rhythm for the next minute, then second-degree block followed by marked PR prolongation and a variety of escape rhythms from progressively further down the conduction system at progressively slower rates. Slight improvement in contractility was never sufficient to provide adequate perfusion and was followed by death after 14 min.Continuous pre- and post-injection ECHO and ECG monitoring were performed to identify the hemodynamic and ECG effects of the total venom or isolated porin. Chironex venom also exhibited profound decreases in ventricular contraction, but rebounds in function were observed subsequent to periods of electromechanical dissociation. In all cases, the progression of electrical abnormalities and contractile dysfunction were delayed compared to untreated mice.Lower doses of venom showed a more varied set of ECG changes that also included markedly reduced contractility. Common ECG changes included a decrease in P wave and varying degrees of atrioventricular block and escape rhythms. Mice injected with zinc gluconate prior to in vitro and in vivo models. These improvements allowed the discovery and elucidation of a sequential release of potassium prior to hemolysis. The finding that this was porin mediated prompted us to test novel approaches with the finding and subsequent development of a novel zinc-based therapy that is superior to the existing antivenom.The absence of effective therapies to reduce fatalities from cubozoan envenomation prompted us to examine the underlying pathophysiology. This required optimization or improvement of venom preparation methodologies, activity bioassays, and animal models of envenomation to better recapitulate authentic envenomation syndromes. One outcome of this comprehensive effort has been the development of a venom preparation method that greatly enhanced total nematocyst content recovery and specific activity. This “total venom” preparation is robust and stable. The development of a reproducible standardized unit of activity (U/mL/%) also allows corollary dose response examination between The venom dose delivered to a victim is related to the number of successfully impaling nematocysts along the tentacle contact site. Various technical limitations in venom preparation have slowed progress in elucidating the mechanism of action of these enigmatic marine venoms. Unlike snake envenomations of milliliter volumes, or cone snail envenomations involving hundreds of microliters, cnidarian envenomations involve the penetration of prey by hundreds of thousands of microscopic specialized penetrant cnidae, or nematocysts, each containing only picoliters of venom. Aqueous extract preparations of whole tentacle, or partially purified cnidae, yield only an incomplete fraction of aqueous soluble venom components that are variously contaminated with other tentacular and structural components, such as cnidae capsule- wall collagens.We report an improved technique that maximizes recovery of intact cnidae from tentacles . Furthermore, this technique allows the full rupture of approximately 90% of these recovered cnidae. Finally, the entire content of the ruptured cnidae, not just the aqueous soluble constituents, were then recovered without the contaminating structural proteins that comprise the nematocyst capsules and tubules. We describe this preparation as “total venom”. The detailed total venom preparation technique described in the Methods section represents an optimization of each step in the isolation of undischarged cnidae free of tentacular contaminants and rupture of the majority of the remarkably robust nematocysts.Comparative analyses of our newly developed method with five other published methodologies in vivo model with the clinically observed sequelae of authentic envenomation. We found Chironex fleckeri venom from tentacular cell-free cnidae to be a complex mixture of proteins, lipids and small bioactive molecules. All cubozoan venoms analyzed to date contain potent hemolytic porins (hemolysins) that share predicted protein structures with a class of self-assembling bacterial pore-forming toxins (PFT), such as anthrolysin O and streptolysin O, which disrupt the permeability barrier of the cell membrane Chironex fleckeri venom exposure in vitro and in vivo after exposure to Chironex fleckeri venom as compared with Alatina moseri. We also observed greater heterogeneity in the ultrastructure of RBC pores formed after exposure to purified porin fractions from Chironex fleckeri versus Alatina moseri. These findings may shed light on the long-standing clinical conundrum that a lack of profound hemolysis in Chironex fleckeri mortality would appear to discount the role of the porin or hemolysin in morbidity and mortality.These critical advances in the recovery of pure and highly active venom allowed us to demonstrate good correlation of our Studies of venom-injected C57BL/6 mice showed rapid and progressive contractile dysfunction with ECG findings consistent with hyperkalemia. The time course of plasma potassium and hemoglobin measurements demonstrated that a catastrophic hyperkalemic state precedes substantial hemolysis. ECGs of mice injected with purified hemolysin showed identical responses to total venom, suggesting that these effects can be specifically attributed to the hemolysin.in vivo toxicities. Zinc gluconate markedly reduced both potassium and hemoglobin efflux from RBC in vitro and delayed mortality in vivo.Previously, investigators have shown that zinc could block bacterial PFT activities Taken together these data suggest that plasma potassium likely originates from porin-perforated RBC. Elevated plasma potassium alone could cause ventricular hypocontractility, and synchronous ECG, as well as electrode measurements, support the role of hyperkalemia. The zinc gluconate-treated mice exhibited a significantly delayed mortality and maintained normal ECG for longer periods following venom or porin exposure. Interestingly, the injection of zinc prior to the administration of venom enhanced mean survival time less than administration of zinc after the venom injection. While the mechanistic basis for this is not immediately clear, bioavailability of plasma zinc may be rapidly reduced by binding to plasma proteins such that available zinc be lower when provided prior to venom injection. The finding that the historic therapeutic approach of antivenom did not enhance survival rates, but rather led to a slight reduction in survival is also noteworthy. Future studies could investigate administration of antivenom following venom injection, as well as combinatorial approaches using both antivenom and zinc compounds.If terminal cardiac events could be delayed in human sting victims, this might provide the opportunity for resuscitative measures that would lower potassium and prevent death. Larger doses of zinc gluconate, or sustained treatment, might be even more effective, for example with a continuous intravenous delivery of zinc ions. Concomitant therapy directed towards immediately reducing hyperkalemia could also prove useful. It is conceivable that topical application of zinc compounds, by inhibiting some of the toxin at the dermal interface, could ameliorate the morbidity of these stings as well. Further studies are warranted to recapitulate these findings in larger animals with cardiovascular physiology (and electrophysiology) closer to humans (such as pigs). Zinc compounds, which are inexpensive, stable and non-toxic at the required dose, could become a useful clinical treatment for potentially lethal cubozoan stings.


MPS, CACS, cIMT, pericardial fat volume, and cardiovascular risk scores were measured.105 consecutive HIV patients  despite similar risk scores. Of HIV patients with perfusion defects, 42% had a CACS = 0. CACS and cIMT were similar in HIV patients and controls. HIV patients on average had 35% increased pericardial fat volume and increased concentration of biomarkers of atherosclerosis in the blood. HIV patients with myocardial perfusion defects had increased pericardial fat volume compared with HIV patients without perfusion defects .HIV patients had an increased prevalence of silent IHD compared to controls as demonstrated by MPS. The finding was strongly associated with pericardial fat volume, whereas cardiovascular risk scores, cIMT and CACS seem less useful as screening tools for detection of myocardial perfusion defects in HIV patients. Since the introduction of combination antiretroviral therapy (cART), mortality has decreased substantially in the HIV infected population Since effective treatments are available to reduce the risk of CVD in patients at risk, early identification of subclinical coronary atherosclerosis is of importance. However, the probable different mechanisms behind development of CVD in HIV patients may limit the usefulness of standard screening tools for CVD.Myocardial perfusion defects demonstrated by myocardial perfusion scintigraphy (MPS) are one of the most prognostic measures that predict cardiovascular events and death in the general population In the general population carotid intima-media thickness (cIMT) correlates well with the presence of ischemic heart disease (IHD) and screening by coronary artery calcium score (CACS) is an accepted measure of atherosclerotic burden The aim of our study was to determine the prevalence of asymptomatic IHD in HIV patients well-managed on cART by means of MPS. In addition, all HIV patients and controls were screened by CACS, cIMT, pericardial fat volume and biomarkers of cardiovascular risk in order to elucidate which screening methods for CVD could be used in HIV infected patients.Written informed consent was obtained from all participants and the study was approved by the Scientific Ethical Committee of the Capital Region of Denmark (KF H-C-2008-060).A total of 105 consecutive HIV patients were prospectively recruited at routine visits at the outpatient clinic at Department of Infectious Diseases, Hvidovre University Hospital between September 2008 and July 2010. Inclusion criteria were i) HIV infected, ii) age 18–70 years and iii) receiving cART>12 months. Exclusion criteria were i) any symptoms or signs suggesting ischemic heart disease. ii) pregnancy, risk of pregnancy or lactation, iii) known heart disease, iv) statin treatment, v) thyrotoxicosis, vi) allergy to contrast media, vii) renal impairment or viii) claustrophobia. However, no subjects were excluded on basis of statin treatment or renal impairment.Every HIV patient, a control subject was matched 1∶1 for age, gender and smoking status. This was done in the following way: for each HIV patient 3 age, gender and smoking matched controls were identified in the Copenhagen City Heart Study database All patients and control subjects had an MPS, CACS, pericardial fat volume measurement, cIMT measurement and a blood sample analysis performed after inclusion in the trial.MPS was conducted in accordance with procedural guidelines of the European Association of Nuclear Medicine. Stress testing was preferentially performed with bicycle ergometer exercise using a standard Bruce protocol . Stress studies were obtained in all patients. Images were acquired 60 minutes after tracer injection of 600–800 MBq of Tc-99 m sestamibi. Gated single photon emission computed tomography myocardial perfusion images were obtained with a Philips Precedence 16P SPECT/CT scanner . A simultaneous CT was used for attenuation correction. Myocardial perfusion images were analyzed using Cedars-Sinai Autoquant 7.2 . All examinations were analyzed by very experienced readers, blinded for HIV status. All examinations were adjudicated as normal or with significant perfusion defect as a consensus reading between two readers. This adjudication was based on all available information; scintigrams with and without attenuation correction, extent and position of any defect, left ventricular volumes and regional wall motion abnormalities.CAC scanning was performed using a Philips Precedence 16P SPECT/CT scanner . Quantification of CACS was done ad modum Agatston as previously described Pericardial fat volume was measured on the previously obtained CT-images of the heart. A region of interest was traced on the boundaries of the pericardial layers in every 10 mm. Volume analysis software  was used to discern fat from other tissues with a threshold of −190 to −30 Hounsfield units. The volume was the sum of all voxels containing fat. Pericardial fat includes both epicardial (located within the pericardium) and paracardial fat  cIMT was determined using a high-resolution ultrasound Doppler system 128 XP/10c  with a linear 7–10 MHz transducer and automatic IMT-measuring tool of image evaluation software , as previously described Plasma lipids and intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), matrix metallopeptidase 9 (MMP9), tissue-type plasminogen activator inhibitor-1 (tPAI-1) and high-sensitivity C-reactive protein (hs-CRP) were measured as previously described The 10-year risk of cardiovascular events (Framingham risk score) With a sample size of 90 matched pairs, a power of 80% for detection of a difference in prevalence of MPD of 8% vs. 0% in the two groups at an alpha = 0.05 was obtained in our one-to-one matched design. Data are presented as mean ± standard error of mean. For comparison between HIV patients and matched controls, categorical variables were compared with related samples McNemar's test and means of continuous variables were compared with paired t-tests. Adjusted p-values for continous variables were calculated using a random effects linear regression model. For comparison between HIV patients with and without perfusion defects, two sample t-test or Fisher's exact test were used for continuous and categorical variables, respectively. Adjusted p-values were calculated using a linear regression model. Binary logistic regression (with stepwise elimination of least significant covariate to test for independent association) was used to test for associations between test result (presence of perfusion defect) and background and examination parameters. Biomarker concentrations were log transformed prior to statistical analysis to obtain normal distribution but are presented untransformed. In some cases 95% confidence intervals (CI) are stated. Statistical analyses were performed using SPSS analysis software version 20.0 . A p-value <0.05 was considered significant.Basic characteristics of the HIV (n = 105) and control (n = 105) populations are presented in Results of cardiovascular imaging by cIMT, CACS, pericardial fat volume and level of biomarkers are presented in Perfusion defects were observed on MPS in 19 of the 105 HIV patients (18%) as opposed to none in the control group (p<0.001). The mean summed stress score in these patients were 7.8 with a mean extent of 10%. The patients with perfusion defects had a mean left ventricular ejection fraction of 52% with only one patient with an ejection fraction below 40%. Only one patient had a significant decrease in ejection fraction during stress compared to rest (from 51% to 43%). None of the patients had significant ECG changes or symptoms during the stress test performed as a maximal bicycle test (n = 18) or adenosine stress (n = 1). The heart rate reached the target of 85% of maximum pulse in all patients with bicycle stress test.All 19 patients with perfusion defects were men. Nine (47%) had never smoked, 15 (79%) had CACS<100, and 8 (42%) had CACS = 0. Thirteen (68%) had metabolic syndrome.HIV patients with perfusion defects on scintigrams had increased pericardial fat volume and waist circumference compared with HIV patients without defects on scintigrams . No incr2 ; p = 0.034) and metabolic syndrome  were all associated with presence of perfusion defect on MPS but age, smoking, cholesterol, systolic blood pressure og DAD 5 year risk were not. In multivariate analysis including all the above-mentioned parameters with backward elimination, only pericardial fat volume remained significant. The 8 HIV patients with a calcium score of 0 but perfusion defects identified on MPS had increased pericardial fat volume compared both with patients with no perfusion defect and a CACS of 0  as well as with no perfusion defect regardless of CACS . In logistic regression analysis, pericardial fat volume was associated with presence of perfusion defect both when including only patients with a CACS of 0 in the model  and when including all patients without perfusion defect regardless of CACS .In univariate binary logistic regression analysis, pericardial fat volume , waist circumference , body surface area ; BMI ; age ; ART duration ; HIV duration  and plasma glucose  whereas no correlation was found with CD4, CD4 nadir, HIV RNA, blood pressure, cholesterol or smoking. In a multivariate linear regression model with all the above-mentioned factors included and backward elimination of least significant factors only abdominal circumference, BMI and age remained in the final model .In HIV-negative controls univariate linear regression analyses revealed that PFV was associated with abdominal circumference ; BMI ; age ; and smoking  whereas no correlation was found with plasma glucose, blood pressure or cholesterol. In a multivariate linear regression model with all the above-mentioned factors included and backward elimination of least significant factors only abdominal circumference, BMI and age remained in final model .This is the first study to investigate silent ischemic heart disease by means of MPS in a group of optimally treated HIV patients and to compare it with an exact 1∶1 matched control group. The major finding was an increased prevalence of myocardial perfusion defects in HIV patients of 18% (95% CI: 12–27%) compared to 0% (95% CI: 0–3%) in HIV-negative controls.Since an abnormal MPS is highly predictive of both fatal and non-fatal myocardial infarction per se but is just a marker of plaque burden. In accordance with this idea, we did not find any clear association between CACS and presence of perfusion defects in HIV patients. Indeed, 42% (8 of 19) of the HIV patients with perfusion defects had a CACS of 0. In addition, the distribution of CACS in both controls and HIV patients were low and as predicted in normal subjects with an FRS of less than 5% The fact that 18% of the HIV patients, all without known CVD, were found to have perfusion defects is potentially important, since intensive prophylactic treatment in these subjects may lead to fewer cardiovascular events. Accordingly, identification of valid screening tools was also a key aim of our study. Although much is known on how to screen the general population, the different pathogenesis in HIV patients is likely to make general screening algorithms less useful. In line with this, the applicability of Framingham risk score has been shown to be less predictive of cardiovascular event in HIV patients Measurement of cIMT is associated with myocardial infarction and coronary artery disease in the general population Thus, some of traditional screening tools in our study seem less useful in identifying HIV patients with myocardial perfusion defects.Changes in fat metabolism and distribution occur and lipodystrophy is present in many HIV patients. Therefore, we also studied fat distribution both as abdominal circumference and by quantifying pericardial fat volume on CT since that latter has been associated with non-calcified plaque burden in HIV negatives This study is limited by the cross-sectional design making it impossible to predict the prognostic consequences of the positive MPS in these asymptomatic patients. Further, this study was not powered to detect possible effect of antiretroviral agents such as abacavir.In conclusion, we observed that HIV patients have a high prevalence of silent ischemic heart disease that is not explainable by traditional risk factors but is strongly associated with pericardial fat volume. Prospective randomized studies should be undertaken to study the potential value identification and prophylactic treatment of these patients.


Adoptive immunotherapy using TCR gene modified T cells may allow separation of beneficial Graft versus tumour responses from harmful GvHD. Improvements to this include methods to generate high avidity or high affinity TCR, improvements in vector design and reduction in mispairing. Following adoptive transfer, TCR transduced T cells must be able to survive and persist in vivo to give most effective antitumour responses. Central memory or naive T cells have both been shown to be more effective than effector cells at expanding and persisting in vivo. Lymphodepletion may enhance persistence of transferred T cell populations. TCR gene transfer can be used to redirect CD4 helper T cells, and these could be used in combination with CD8+ tumour specific T cells to provide help for the antitumour response. Antigen specific T regulatory T cells can also be generated by TCR gene transfer and could be used to suppress unwanted alloresponses. Allogeneic haematopoietic stem cell transplantation (HSCT) is an effective treatment for many haematological malignancies. In addition, unselected donor lymphocyte infusions (DLIs) can be utilized to successfully treat relapsed leukaemia after HSCT . DependiRefining the concept of donor lymphocyte infusions by isolating donor lymphocytes that have known tumour reactivity may result in more effective GvT. Falkenburg et al. have utilized donor-derived leukaemia reactive cytotoxic T lymphocytes (CTLs) to treat a patient with relapsed accelerated phase CML after HSCT . The patAs an alternative to isolating tumour reactive lymphocytes, the specificity of T cells can be redirected by retroviral gene transfer of T-cell receptor (TCR) genes. This allows T cells to be generated that are specific for a defined tumour antigen presented by MHC. TCR gene transfer using retroviruses was first demonstrated by Clay et al. who transduced human T cells with a TCR that was specific for a melanoma antigen presented by HLA-A2. These redirected T cells had demonstrable antitumour reactivity in vitro . TCRs taThe first clinical trial using TCR gene modified T cells was for treatment of patients with metastatic melanoma. In this study, autologous peripheral blood lymphocytes were transduced with alpha and beta chains specific for a melanoma TAA, MART-1, and adoptively transferred to a cohort of patients with metastatic melanoma. This resulted in an objective response rate in 2/15 patients 13%) with both responders obtaining long-term disease remission % with bo. TCR-traImproving the therapeutic GvT effect may be achieved by an increase in the functional avidity of the transduced T cell for its specific tumour antigen. Increasing T cell avidity has been correlated with improved elimination of tumour cells in vivo . AvidityOne method to generate high-avidity TAA-specific CTLs is to immunize human HLA expressing transgenic mice with human TAA peptides thus eliciting an immune response against the human peptide. CTLs that express high-avidity TCRs can then be selected, and the TCR genes isolated and used to transduce peripheral human lymphocytes , 19. Altμmol [Naturally occurring TCRs are typically of low affinity, normally within the range of 1–100 μmol , 23. TCRμmol –26. Thisμmol . Zhao etμmol . One othμmol .Higher surface expression of TCR has been shown to correlate with an increased responsiveness to specific antigen –31. ImprWhen introducing a new alpha and beta chain into a T cell, ideally they will pair only with one another. However, the introduced alpha and beta chains may form mixed heterodimers whereby the introduced alpha chain pairs with the endogenous beta chain and the introduced beta chain pairs with the endogenous alpha chain. This can lead to the expression of 2 new TCR specificities by the T cell with the potential to target normal tissues as they will not have undergone selection in the thymus. In addition, this will lead to lower expression of the correctly paired TCR and thus lower the functional avidity of the T cell. There are now a number of methods to reduce mispairing. The introduction of a new disulphide bond into the constant region of the introduced alpha and beta chain ensures that these chains will more efficiently bond with each other and not with the endogenous TCR chains. This has been shown to reduce mispairing and improve the functional avidity of T cells –38. ReplTCR gene transfer experiments have demonstrated that TCR cell surface composition following transduction is dependent on properties of both the introduced TCR and the existing endogenous TCR. This has been described as TCR strength, with “strong” TCRs being more efficiently expressed on the cell surface compared to “weak” TCRs. T-cell strength may be controlled by the intrinsic pairing properties of the alpha and beta chains and also by their ability to bind to CD3 . The intThe ability of transduced T cells to persist long-term, mount robust memory responses and migrate to the site of response following adoptive transfer is key to successful antitumour responses. Persistence of transferred tumour reactive T cells has been shown to correlate with an effective therapeutic effect in adoptive immunotherapy utilizing TIL for treatment of metastatic melanoma . From thNaive, central memory (CM) and effector memory (EM) populations each have distinct phenotypic and functional characteristics. In vitro stimulation of these subsets induces their proliferation and differentiation into cytolytic effector cells. EM T cells preferentially home to peripheral tissues, have immediate effector function on antigen rechallenge but show only poor numeric expansion. They have downregulated CD62L although maintain expression of CCR7. CM T cells have high expression of CD62L and CCR7 and thus can home to and recirculate through secondary lymphoid organs where they can interact efficiently with antigen-presenting cells. They have enhanced proliferative capacity upon antigen reencounter , 46. A cBerger et al. looked at the persistence of EM T cells versus CM T cells following adoptive transfer of CD8+ CMV-specific effector cells into unconditioned primates. The effector T cells that were derived from EM T cells only persisted in the blood for a short period and were unable to persist within lymph node or bone marrow or peripheral tissues. They were also unable to differentiate back into an EM T-cell phenotype. In contrast the effector T cells derived from CM T-cells were able to persist in blood and could migrate to bone marrow and lymph nodes and had the ability to differentiate to both CM and EM T cell phenotype. Effector cells derived from CM T cells appeared to be more plastic than EM T cells, retaining the ability to revert to both a CD62L− and CD62+ memory cell phenotype .γ and IL-2 following ex vivo restimulation [However, this study did not address the role of naive T cells for use in adoptive immunotherapy. Hinrichs et al. utilizing a murine adoptive transfer model have suggested that naive T cells may be superior to T CM cells. T cells were isolated from pmel-1 TCR-transgenic mice which express TCR specific for self-antigen, gp100 that is also expressed by murine melanoma cells. Sublethally irradiated mice with established B16 melanoma tumours received adoptive transfer of CD8+ naive T cells (CD62L+ CD44low) or CM T cells (CD62L+CD44high). All mice also received specific peptide vaccination and IL-2. Both naive and CM T cells were able to mediate tumour regression following adoptive transfer, but effector cells derived from naive T cells had superior antitumour activity. Adoptively transferred naive T cells also showed greater expansion within spleen and draining LN and also produced higher amounts of IFN-mulation .β(Gsk-3β). Gsk-3β inhibitors lead to an accumulation of beta catenin which mimics Wnt signaling [β inhibitors. This inhibited cells from acquiring a T effector phenotype and also generated a CD44 low CD62L high T-cell population which had properties of stem cells, that is, robust self-renewal and the capacity to generate CM, EM, and effector T cells. They also had a characteristic phenotype, with high expression of Sca1, CD122, and Bcl2, previously described by Zhang et al. as CD8 memory stem cells [A novel way to try to prevent T cells acquiring a T effector phenotype prior to adoptive transfer is to target the Wnt/Beta Catenin pathway which is thought to be important in the generation and maintenance of T-cell memory . The Wntignaling . Utiliziem cells . Followiem cells . TargetiLymphodepletion as a result of conditioning therapy preadoptive transfer has been shown to increase persistence of transferred T cells. Trials in melanoma patients conditioned with chemotherapy or chemotherapy plus total body irradiation demonstrated that inducing lymphopaenia improved the persistence of the transferred T-cell population , 41, 52.Most adoptive immunotherapy protocols have focused on the transfer of CD8+ tumour-specific T cells. However, in the absence of CD4 help, CD8+ T cells have impaired function, persistence, and cannot mount effective memory responses –68. The Quezada et al. explored the role of classical class II-restricted CD4+ T cells in a murine model of advanced melanoma. Adoptive transfer of small numbers of naive class II-restricted tumour specific CD4+ T cells into irradiated recipients led to in vivo differentiation and expansion of CD4+ T cells and tumour regression. These CD4+ T cells acquired cytotoxic activity and directly rejected the tumour in a class II-dependent manner. The efficacy of CD4+ T cells was increased by concurrent blockade of CTLA-4 resulting in greater expansion of CD4+ T cells and decreased numbers of T regulatory cells .γ response. In vivo, CD4+ T cells could provide help for CTL-mediated tumour eradication, and these cells persisted in vivo for up to 90 days after tumour regression and were able to reexpand following tumour challenge. The generation of class I-restricted TCRs is a promising strategy with the advantage that tumour-specific CD4+ and CD8+ T cells targeting the same epitope can be generated.The use of CD4 + T cells for adoptive immunotherapy is limited due to a lack of well-characterized tumour antigens presented by class II MHC. In addition the majority of tumour cells are class II negative and therefore cannot directly present antigen to CD4+ T cells. To overcome these limitations, CD4+ T cells expressing class I restricted TCR have been generated by gene transfer. It has been demonstrated that CD4 T cells transduced with class I-restricted TCR can provide antigen-specific helper functions. Some class I-restricted TCRs are independent of the CD8 coreceptor and function in CD4+ T cells in the absence of additional CD8 . HoweverCD4+ CD25+ FOXP 3+ T regulatory cells (CD4+ T regs) play a crucial role in the control of GvHD. This has been demonstrated via the depletion of CD4+ T regs from donor cells in murine MHC-mismatched transplant models. These transplants were done using a variety of different strain combinations, and the depletion of CD4+ T regs resulted in an increase in GVHD and an increase in mortality . AdoptivThe use of antigen-specific CD4+ T regs is more effective at reducing GvHD than a polyclonal population . CD4+ T Gene transfer of antigen-specific CD4+ T cells with FOXP3 is one possible mechanism of generating large numbers of antigen-specific CD4+ T regs. The ectopic expression of FOXP3 had been shown to convert CD4+ T cells to regulatory T cells that are phenotypically and functionally the same as CD4+ T regs. Albert et al. produced CD4+ T regs by transducing CD4+ CD25- TCR-transgenic cells with FOXP3 and found that FOXP3-transduced T regs were as effective as wild-type CD4+ T regs at suppressing GvHD . AntigenIn order to develop effective adoptive immunotherapy protocols, it is important that the correct TAA or epitope is targeted. The use of high throughput screening of cancer genomes can identify large numbers of novel TAA, and it is important that research is focused, leading to more effective translation of TCR gene therapy and vaccine strategies into clinical practice. An ideal tumour antigen would be one that is widely expressed in different tumour types and not unique to individual patients. Targeting tumour antigens that are essential to the oncogenic process or cancer cell survival may induce sustained tumour regression. Specifically targeting a single epitope, however, may lead to the selection of tumour variants that lack the target antigen as a result of antigen loss or aberrant presentation via MHC loss –83. SeleA National Cancer Institute Pilot project has been developed to try to prioritize antigens for further research and development . This utIn addition to reducing the efficacy of TCR transduced T cells, mispairing is also a concern with regards to safety of TCR-transferred T cells for clinical use. Bendle et al. have demonstrated that TCR-transduced T cells can induce GVHD following adoptive transfer. This is not a result of the on-target toxicity of the introduced TCR but is a result of the formation of new heterodimers that have off-target toxicity directed against normal tissues . This stLymphocytes can be transduced with suicide genes which enables selective elimination of transduced cells should GvHD develop following adoptive transfer. The most extensively studied to date is herpes simplex thymidine kinase (HSV-TK). HSV-TK phosphorylates ganciclovir to a toxic metabolite which interrupts DNA elongation resulting in the selective death of cells transduced with HSV-TK following the infusion of ganciclovir. When administered to patients with relapsed haematological malignancy after allogeneic HLA-matched HSCT, rates of GvT using HSV-TK-transduced lymphocytes were comparable to that of DLI using unmanipulated lymphocytes. The administration of ganciclovir for treatment of GVHD or for CMV reactivation selectively eliminated HSV-TK-transduced cells and improved GvHD . HSV-TK-Another suicide gene strategy in development is based on human apoptosis proteins. A fusion protein consisting of a late stage apoptosis molecule, caspase 9 fused to a FK506-binding protein (FK506BP) analogue has been transduced into lymphocytes. Apoptosis of the transduced cells is induced following the administration of a chemical inducer of dimerization (CID) which results in aggregation and activation of caspase 9. The CID which has been used is a nontoxic FK506 analog which has been modified so that it has much higher affinity for the modified FK506BP than the endogenous FK506BP. A single dose of CID resulted in death of >99% of transduced cells both in vitro and in vivo. These molecules are human derived and therefore should in theory be less immunogenic than HSV-TK .γ) chain, and there is some controversy over whether this gene itself had a role as a cooperating oncogene. Insertions near both the LM02 and IL-2R-γ chain gene have been described in murine T-cell leukaemia [γ chain led to a high incidence of lymphoma in a murine model system [γ chain did not have any effect on haematopoietic cell development whilst overexpression of LMO2 led to a block in T-cell development leading to a preleukaemic phenotype [γ chain was not sufficient to lead to induction of leukaemia following HSCT in mouse models using both lentiviral and retroviral vectors [ An additional safety concern of gene therapy is insertional mutagenesis secondary to gene insertion into the host chromosome leading to disruption or activation of cellular genes. Out of the 19 X-linked severe combined immunodeficiency patients treated with retrovirally transduced haematopoietic stem cells there have been 5 cases of T-cell leukaemia reported –94. In 4eukaemia . In addil system . Pike-Ovhenotype . Modlich vectors .All cases of leukaemia arising secondary to insertional mutagenesis have involved the use of retroviral vectors which have LTRs with strong enhancer/promoters elements. Strong LTRs may cause the transcription of cellular genes in addition to the transgene which can lead to unregulated gene expression. By using self inactivating vectors (SIN), it may be possible to reduce the risk of insertional mutagenesis. SIN vectors contain a deletion within the U3 region of the 3′LTR which leads to inactivation of the 5′LTR following reverse transcription, thus, inactivating both LTRs [oth LTRs , 100. Anoth LTRs –103.Both lentiviral and gamma retroviral vectors integrate in a semirandom fashion with a bias towards insertion into transcriptional units. Gamma retroviral vectors tend to integrate close to transcription start sites or DNA regulatory areas such as CpG islands. Insertions of viral LTR at these points will have a higher probability of causing aberrant gene expression , 105. InThe risk of insertional mutagenesis may also be affected by the cell type being transduced, and there is a lower risk of transformation by transducing terminally differentiated cells as opposed to HSC. A study looking at long-term followup of patients receiving T cells transduced with thymidine kinase genes in the context of HSCT demonstrated that up to 20% of gene insertions resulted in altered gene expression of neighbouring genes, but there had been no incidence of clonal selection in these cells . To dateTCR gene transfer to produce antigen-specific T cells represents a targeted approach to treatment of haematological malignancies which may generate more specific GvT responses whilst reducing harmful GvHD responses. In addition to using TCR-transduced T cells to enhance GvT, we can also generate antigen-specific T regs to directly suppress harmful GVHD responses. Experience from clinical trials of melanoma antigen-specific TCR-transduced T cells has highlighted areas where this technique can be further refined and improved. Improvements in vector design, generation of high-avidity TCR, and reduction of TCR mispairing has all led to improvements in preclinical models. The importance of phenotype and subtype of the transferred population and the host environment into which they are transferred is becoming increasingly evident. Clinical trials utilizing WT1 TCR-transduced lymphocytes for treatment of haematological malignancies are due to commence in the near future hopefully leading to progress translating this technique into wider clinical use.