Generation of functional CD8+ T Cells by human dendritic cells expressing glypican-3 epitopes

<p>Abstract</p> <p>Background</p> <p>Glypican 3 (GPC-3) is an oncofoetal protein that is expressed in most hepatocellular carcinomas (HCC). Since it is a potential target for T cell immunotherapy, we investigated the generation of functional, GPC-3 specific T cells from peripheral blood mononuclear cells (PBMC).</p> <p>Methods</p> <p>Dendritic cells (DC) were derived from adherent PBMC cultured at 37°C for 7 days in X-Vivo, 1% autologous plasma, and 800 u/ml GM-CSF plus 500 u/ml IL-4. Immature DC were transfected with 20 μg of <it>in vitro </it>synthesised GPC-3 mRNA by electroporation using the Easy-ject plus system (Equibio, UK) (300 V, 150 μF and 4 ms pulse time), or pulsed with peptide, and subsequently matured with lipopolysaccharide (LPS). Six predicted GPC-3 peptide epitopes were synthesized using standard f-moc technology and tested for their binding affinity to HLA-A2.1 molecules using the cell line T2.</p> <p>Results</p> <p>DC transfected with GPC-3 mRNA but not control DC demonstrated strong intracellular staining for GPC-3 and <it>in vitro </it>generated interferon-gamma expressing T cells from autologous PBMC harvested from normal subjects. One peptide, GPC-3_522-530FLAELAYDL, fulfilled our criteria as a naturally processed, HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope: i) it showed high affinity binding to HLA-A2, in T2 cell binding assay; ii) it was generated by the MHC class I processing pathway in DC transfected with GPC-3 mRNA, and iii) HLA-A2 positive DC loaded with the peptide stimulated proliferation in autologous T cells and generated CTL that lysed HLA-A2 and GPC-3 positive target cells.</p> <p>Conclusions</p> <p>These findings demonstrate that electroporation of GPC-3 mRNA is an efficient method to load human monocyte-derived DC with antigen because <it>in vitro </it>they generated GPC-3-reactive T cells that were functional, as shown by interferon-gamma production. Furthermore, this study identified a novel naturally processed, HLA-A2-restricted CTL epitope, GPC-3_522-530FLAELAYDL, which can be used to monitor HLA-A2-restricted CTL responses in patients with HCC. Further studies are required to investigate whether anti-GPC-3 immunotherapy has a role in the treatment of GPC-3 dependent tumours, such as HCC.</p

Putative Auditory-Evoked Neurophonic Measurements Using a Novel Signal Processing Technique: A Pilot Case Study

With changes to cochlear implant candidacy and improvements in surgical technique, there is a need for accurate intraoperative assessment of low-frequency hearing thresholds during cochlear implantation. In electrocochleography, onset compound action potentials (CAPs) typically allow estimation of auditory threshold for frequencies above 1 kHz, but they are less accurate at lower frequencies. Auditory nerve neurophonic (ANN) waveforms, on the other hand, may overcome this limitation by allowing phase-locked neural activity to be tracked during a prolonged low-frequency stimulus rather than just at its onset (Henry, 1995). Lichtenhan et al. (2013) have used their auditory nerve overlapped waveform (ANOW) technique to measure these potentials from the round windows of cats and guinea pigs, and reported that in guinea pigs these potentials originate in the cochlear apex for stimuli below 70 dB SPL (Lichtenhan et al., 2014). Human intraoperative round window neurophonic measurements have been reported by Choudhury et al. (2012). We have done the same in hearing impaired awake participants, and present here the results of a pilot study in which we recorded responses evoked by 360, 525, and 725 Hz tone bursts from the cochlear promontory of one participant. We also present a modification to the existing measurement technique which halves recording time, extracting the auditory neurophonic by recording a single averaged waveform, and then subtracting from it a 180° group-delayed version of itself, rather than using alternating condensation and rarefaction sound stimuli. We cannot conclude that the waveforms we measured were purely neural responses originating from the apex of the cochlea: as with all neurophonic measurement procedures, the neural responses of interest cannot be separated from higher harmonics of the cochlear microphonic without forward masking, regardless of electrode location, stimuli or post-processing algorithm. In conclusion, the extraction of putative neurophonic waveforms can easily be incorporated into existing electrocochleographic measurement paradigms, but at this stage such measurements should be interpreted with caution

Twin pregnancy in a liver transplant recipient with HIV infection

We are not aware of a report detailing the complex obstetrical and medical management of twin pregnancy in the context of HIV infection and early post-liver transplantation period. Here we describe the successful outcome of a twin pregnancy in a 28-year-old HIV-positive female receiving antiretroviral therapy and immunosuppressive therapy who was the recipient of a liver transplant for previous drug-induced liver failure

Development and validation of a mathematical equation to estimate glomerular filtration rate in cirrhosis: The rfh cirrhosis Gfr

Current expressions based on serum creatinine concentration overestimate kidney function in cirrhosis leading to significant differences between "true" and calculated glomerular filtration rate (GFR). We compared the performance of MDRD-4, MDRD-6 and CKD-EPI with "true" GFR and the impact of this difference on MELD calculation. We subsequently developed and validated a GFR equation specifically for cirrhosis and compared the performance of the new derived formula with existing GFR formulas. We included 469 consecutive patients who had a transplant assessment between 2011 and 2014. "True" GFR (mGFR) was measured using plasma isotope clearance according to a technique validated in patients with ascites. A corrected creatinine was derived from the mGFR after application of the MDRD formula. Subsequently, a corrected MELD was calculated and was compared with the conventionally calculated MELD. Stepwise multiple linear regression was used to derive a GFR equation. This was compared with the measured GFR in independent external and internal validation sets of 82 and 174 patients with cirrhosis respectively. A difference>20 ml/min/1.73m(2) between existing formulae and mGFR was observed in 226 (48.2%) patients. The corrected MELD score was ≥3 points higher in 177 (37.7%) patients. The predicted equation derived (R(2) =74·6%) was: GFR=45·9x(creatinine(-0) ·(836) )x(urea(-0) ·(229) )x(INR(-0) ·(113) )x(age(0) ·(129) )x(sodium(0) ·(972) )x1·236(if male)x0·92(if moderate/severe ascites). The model was a good fit and showed the greatest accuracy compared to that of existing formulae. CONCLUSION: We developed and validated a new accurate model for GFR assessment in cirrhosis, the RFH cirrhosis GFR, using readily available variables. This remains to be tested and incorporated in prognostic scores in patients with cirrhosis

Bacterial Infections Change Natural History of Cirrhosis Irrespective of Liver Disease Severity

OBJECTIVES: We assessed the prognostic significance of infections in relation to current prognostic scores and explored if infection could be considered per se a distinct clinical stage in the natural history of cirrhosis. METHODS: We included consecutive patients with cirrhosis admitted to a tertiary referral liver unit for at least 48 h over a 2-year period. Diagnosis of infection was based on positive cultures or strict established criteria. We used competing risk analysis and propensity score matching for data analysis. RESULTS: 501 patients (63% male, 48% alcoholic liver disease, median Model of End-stage Liver Disease (MELD)=17) underwent 781 admissions over the study period. Portal hypertensive bleeding and complicated ascites were the commonest reasons of admission. The incidence of proven bacterial infection was 25.6% (60% community acquired and 40% nosocomial). Survival rates at 3, 6, 12, and 30 months were 83%, 77%, 71%, and 62% in patients without diagnosis of infection, vs. 50%, 46%, 41%, and 34% in patients with diagnosis of infection. Overall survival was independently associated with MELD score (hazards ratio (HR) 1.099), intensive care (ITU) stay (HR 1.967) and bacterial infection (HR 2.226). Bacterial infection was an independent predictor of survival even when patients who died within the first 30 days were excluded from the analysis in Cox regression (HR 2.013) and competing risk Cox models in all patients (HR 1.46) and propensity risk score-matched infected and non-infected patients (HR 1.67). CONCLUSIONS: Infection most likely represents a distinct prognostic stage of cirrhosis, which affects survival irrespective of disease severity, even after recovery from the infective episode

Elevated liver enzymes in inflammatory bowel disease: the role and safety of infliximab

BACKGROUND: Abnormal liver enzymes are frequently encountered in inflammatory bowel disease (IBD) patients. Infliximab has been implicated in inducing drug-induced liver injury, autoimmune hepatitis or reactivation of hepatitis B virus. We aimed to clarify the role of infliximab in liver impairment in an IBD cohort. STUDY: A total of 305 patients with IBD, without evidence of chronic liver disease, were included in the study and retrospectively evaluated. Laboratory and clinical data were retrieved from a prospectively acquired database. In all, 176 consecutive patients treated with infliximab during the last 5 years were compared with a matched population of 129 patients who did not receive any antitumour necrosis factor treatment. RESULTS: Elevation of alanine transaminase (ALT) was frequent in the entire population (36.4%) and it was not significantly associated with the use of infliximab (P=0.284). Elevations more than 3 upper limit of normal were observed in 7.9% and these resolved spontaneously in 83%. The use of immunomodulators was the only factor that was significantly associated with liver enzyme abnormalities in multivariate analysis [odds ratio (OR) 2.666, 95% confidence interval (CI) 1.576-4.511, P<0.005]. Overall, 39% of patients on infliximab had elevated liver enzymes and this was associated with increased ALT before starting infliximab (OR 3.854, 95% CI 1.800-8.251, P=0.001) and with longer duration of infliximab treatment (OR 1.030, 95% CI 1.013-1.047, P=0.001). CONCLUSION: Elevated liver enzymes are frequently found in IBD patients and they usually resolve spontaneously. The use of immunomodulators was independently associated with increased ALT. Infliximab is relatively safe in terms of liver impairment and discontinuation of treatment is rarely required in the setting of modest elevations of ALT

Albuterol metered dose inhaler performance under hyperbaric pressures

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionINTRODUCTION: The stimulus for this presentation was an asthma attack suffered on the first dive by a victim of a severe industrial electrical burn. The patient's response to albuterol metered dose inhaler (MDI) treatment given at depth was felt to have been poor. We thus wondered what the output of these devises (chlorofluorocarbon or CFC) was at therapeutic depth versus normobaria. As the current MDIs were being phased out of use we also wondered what the comparable output characteristics of the replacement MDIs (hydrofluoroalkane or HFA) would be. MATERIALS AND METHODS: The dose and aerosol particle size and number delivered by MDIs were measured in a hyperbaric chamber at pressures ranging from one atmosphere absolute (1 ATA, 0 feet of seawater, fsw, 101 kPa) to three ATA (66 fsw, 304 kPa). Mass delivered was measured by a Sartorius B120 analytical balance, and particle size analysis by a TSI 3080L electrostatic classifier with a TSI 3776 ultrafine condensation particle counter. RESULTS: Dose delivery per actuation by CFC and long canister HFA powered MDIs was 13±1% and 12±1% less, respectively, at 3 ATA compared to 1 ATA. However, dose delivery by short canister HFA MDIs was not significantly changed with pressure. The geometric mean diameters of nano particles from the CFC and short canister HFA MDIs decreased from 50 nm at 0 fsw to 32 nm at 66 fsw whereas the long canister HFA aerosol diameters were not affected. The numbers of nanometer size particles delivered at 66 fsw were only 4-7% of those delivered at 0 fsw for the CFC and long canister HFA MDIs; whereas for the short canister HFAs it was 26%. CONCLUSIONS: The doses of albuterol and the sizes and numbers of aerosol particles emitted from albuterol MDIs actuated in a hyperbaric environment vary by canister type; CFC MDI loss is probably unimportant

The fate of indeterminate liver lesions: What proportion are precursors of hepatocellular carcinoma?

BACKGROUND: The natural history and incidence of hepatocellular carcinoma (HCC) arising from indeterminate liver lesions are not well described. We aimed to define the incidence of HCC in a cohort of patients undergoing surveillance by magnetic resonance imaging (MRI) and estimate any associations with incident HCC. METHODS: We performed a retrospective follow-up study, identifying MRI scans in which indeterminate lesions had been reported between January 2006 and January 2017. Subsequent MRI scan reports were reviewed for incident HCC arising from indeterminate lesions, data were extracted from electronic patient records and survival analysis performed to estimate associations with baseline factors. RESULTS: One hundred and nine patients with indeterminate lesions on MRI were identified. HCC developed in 19 (17%) patients over mean follow up of 4.6 years. Univariate Cox proportional hazards analysis found incident HCC to be significantly associated with baseline low platelet count (hazard ratio (HR) = 7.3 (95% confidence intervals (CI) 2.1-24.9), high serum alpha-fetoprotein level (HR = 2.7 (95% CI 1.0-7.1)) and alcohol consumption above fourteen units weekly (HR = 3.1 (95% CI 1.1-8.7)). Multivariate analysis, however, found that only low platelet count was independently associated with HCC (HR = 5.5 (95% CI 0.6-5.1)). CONCLUSIONS: HCC arises in approximately one fifth of indeterminate liver lesions over 4.6 years and is associated with a low platelet count at the time of first diagnosis of an indeterminate lesion. Incidence of HCC was more common in people with viral hepatitis and in those consuming > 14 units of alcohol per week. Our data may be used to support a strategy of enhanced surveillance in patients with indeterminate lesions