A novel GFP nude rat model to investigate tumor-stroma interactions

The anticancer effects of ceramide have been reported in many types of cancers but less in lung cancer. In this study, we used C₂-ceramide to further investigate its possible anticancer effects and mechanisms on non-small cell lung cancer (NSCLC) H1299 cells. The result of cell proliferation in terms of trypan blue assay showed high dose of C₂-ceramide inhibited cell survival after 24 h treatment. The flow cytometry-based assays indicated the effect of apoptosis, chromatin condensation, and G₁ arrest in terms of Annexin V/propidium iodide (PI), DAPI, and PI stainings, respectively. Moreover, the decreased protein level of p-Akt, p-NFκB, survivin and cyclin A2 were detected by Western blot assay. Taken together, these results indicated the antiproliferative effect of C₂-ceramide is majorly responsible for cell apoptosis in lung cancer H1299 cells.     

177Lu-DOTA-HH1, a Novel Anti-CD37 Radio-Immunoconjugate: A Study of Toxicity in Nude Mice

Background

CD37 is an internalizing B-cell antigen expressed on Non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia cells (CLL). The anti-CD37 monoclonal antibody HH1 was conjugated to the bifunctional chelator p-SCN-Bn-DOTA and labelled with the beta-particle emitting radionuclide ¹⁷⁷Lu creating the radio-immunoconjugate (RIC) ¹⁷⁷Lu-DOTA-HH1 (¹⁷⁷Lu-HH1, trade name Betalutin). The present toxicity study was performed prior to initiation of clinical studieswith ¹⁷⁷Lu-HH1.

Methodology/Principal Findings

Nude mice with or without tumor xenografts were treated with 50 to 1000 MBq/kg ¹⁷⁷Lu- HH1 and followed for clinical signs of toxicity up to ten months. Acute, life threatening bone marrow toxicity was observed in animals receiving 800 and 1000 MBq/kg ¹⁷⁷Lu-HH1. Significant changes in serum concentrations of liver enzymes were evident for treatment with 1000 MBq/kg ¹⁷⁷Lu-HH1. Lymphoid depletion, liver necrosis and atrophy, and interstitial cell hyperplasia of the ovaries were also observed for mice in this dose group.

Conclusions/Significance

¹⁷⁷Lu-DOTA-HH1 was well tolerated at dosages about 10 times above those considered relevant for radioimmunotherapy in patients with B-cell derived malignancies.The toxicity profile was as expected for RICs. Our experimental results have paved the way for clinical evaluation of ¹⁷⁷Lu-HH1 in NHL patients.     

Whole CMV Proteome Pattern Recognition Analysis after HSCT Identifies Unique Epitope Targets Associated with the CMV Status

Cytomegalovirus (CMV) infection represents a vital complication after Hematopoietic Stem Cell Transplantation (HSCT). We screened the entire CMV proteome to visualize the humoral target epitope-focus profile in serum after HSCT. IgG profiling from four patient groups (donor and/or recipient +/− for CMV) was performed at 6, 12 and 24 months after HSCT using microarray slides containing 17174 of 15mer-peptides overlapping by 4 aa covering 214 proteins from CMV. Data were analyzed using maSigPro, PAM and the ‘exclusive recognition analysis (ERA)’ to identify unique CMV epitope responses for each patient group. The ‘exclusive recognition analysis’ of serum epitope patterns segregated best 12 months after HSCT for the D+/R+ group (versus D−/R−). Epitopes were derived from UL123 (IE1), UL99 (pp28), UL32 (pp150), this changed at 24 months to 2 strongly recognized peptides provided from UL123 and UL100. Strongly (IgG) recognized CMV targets elicited also robust cytokine production in T-cells from patients after HSCT defined by intracellular cytokine staining (IL-2, TNF, IFN and IL-17). High-content peptide microarrays allow epitope profiling of entire viral proteomes; this approach can be useful to map relevant targets for diagnostics and therapy in patients with well defined clinical endpoints. Peptide microarray analysis visualizes the breadth of B-cell immune reconstitution after HSCT and provides a useful tool to gauge immune reconstitution.     

Hepatitis C Viremia Patterns in Incident Hepatitis C Infection and One Year Later in 150 Prospectively Tested Persons Who Inject Drugs

Objectives

To assess HCV viremia levels just before, during and one year after anti-HCV seroconversion in people who inject drugs (PWID).

Methods

PWID enrolling into a needle exchange program in Malmö, Sweden, 1997–2005 constituted the source population. Sera were obtained at enrolment and at approximately 3–4 monthly intervals afterwards, and were initially tested for anti-HIV, HBsAg/anti-HBc and anti-HCV and thereafter for markers previously negative. Seroconversion to anti-HCV had occurred during the study period in 186 out of 332 seronegative subjects. In these anti-HCV seroconverters, quantitative HCV RNA PCR was retrospectively performed on frozen sera to determine viremia levels in the last anti-HCV negative, the first anti-HCV positive and in one year follow-up samples.

Results

Among 150 subjects seroconverting to anti-HCV with samples available from all three defined time-points, eight different patterns of viremia were observed. Spontaneous clearance at one year was noted in 48 cases (32%) and was associated with female gender (p = 0.03, CI 0.17–1.00). In 13 cases HCV-RNA was not detected in any study sample. Among 61 subjects with pre-seroconversion viremia, viral load was significantly higher in the pre-seroconversion samples compared to subsequent samples. For the whole group, viral load declined to undetectable levels at seroconversion in 28% of cases (but with recurrent viremia in 15%).

Conclusions

Different patterns of HCV RNA kinetics were observed among PWID with documented seroconversion to anti-HCV. The frequently observed absence of detectable HCV RNA in the first anti-HCV positive sample (irrespective of subsequent viremia) demonstrates the importance of repeated sampling and RNA testing for determination of the outcome of acute infection.     

Size-Based Enrichment of Exfoliated Tumor Cells in Urine Increases the Sensitivity for DNA-Based Detection of Bladder Cancer

Bladder cancer is diagnosed by cystoscopy, a costly and invasive procedure that is associated with patient discomfort. Analysis of tumor-specific markers in DNA from sediments of voided urine has the potential for non-invasive detection of bladder cancer; however, the sensitivity is limited by low fractions and small numbers of tumor cells exfoliated into the urine from low-grade tumors. The purpose of this study was to improve the sensitivity for non-invasive detection of bladder cancer by size-based capture and enrichment of tumor cells in urine. In a split-sample set-up, urine from a consecutive series of patients with primary or recurrent bladder tumors (N = 189) was processed by microfiltration using a membrane filter with a defined pore-size, and sedimentation by centrifugation, respectively. DNA from the samples was analyzed for seven bladder tumor-associated methylation markers using MethyLight and pyrosequencing assays. The fraction of tumor-derived DNA was higher in the filter samples than in the corresponding sediments for all markers (p<0.000001). Across all tumor stages, the number of cases positive for one or more markers was 87% in filter samples compared to 80% in the corresponding sediments. The largest increase in sensitivity was achieved in low-grade Ta tumors, with 82 out of 98 cases positive in the filter samples (84%) versus 74 out of 98 in the sediments (75%). Our results show that pre-analytic processing of voided urine by size-based filtration can increase the sensitivity for DNA-based detection of bladder cancer.     

Identification of a Common Non-Apoptotic Cell Death Mechanism in Hereditary Retinal Degeneration

Cell death in neurodegenerative diseases is often thought to be governed by apoptosis; however, an increasing body of evidence suggests the involvement of alternative cell death mechanisms in neuronal degeneration. We studied retinal neurodegeneration using 10 different animal models, covering all major groups of hereditary human blindness (rd1, rd2, rd10, Cngb1 KO, Rho KO, S334ter, P23H, Cnga3 KO, cpfl1, Rpe65 KO), by investigating metabolic processes relevant for different forms of cell death. We show that apoptosis plays only a minor role in the inherited forms of retinal neurodegeneration studied, where instead, a non-apoptotic degenerative mechanism common to all mutants is of major importance. Hallmark features of this pathway are activation of histone deacetylase, poly-ADP-ribose-polymerase, and calpain, as well as accumulation of cyclic guanosine monophosphate and poly-ADP-ribose. Our work thus demonstrates the prevalence of alternative cell death mechanisms in inherited retinal degeneration and provides a rational basis for the design of mutation-independent treatments.     

Exploring the Human Plasma Proteome for Humoral Mediators of Remote Ischemic Preconditioning - A Word of Caution

Despite major advances in early revascularization techniques, cardiovascular diseases are still the leading cause of death worldwide, and myocardial infarctions contribute heavily to this. Over the past decades, it has become apparent that reperfusion of blood to a previously ischemic area of the heart causes damage in and of itself, and that this ischemia reperfusion induced injury can be reduced by up to 50% by mechanical manipulation of the blood flow to the heart. The recent discovery of remote ischemic preconditioning (RIPC) provides a non-invasive approach of inducing this cardioprotection at a distance. Finding its endogenous mediators and their operative mode is an important step toward increasing the ischemic tolerance. The release of humoral factor(s) upon RIPC was recently demonstrated and several candidate proteins were published as possible mediators of the cardioprotection. Before clinical applicability, these potential biomarkers and their efficiency must be validated, a task made challenging by the large heterogeneity in reported data and results. Here, in an attempt to reproduce and provide more experimental data on these mediators, we conducted an unbiased in-depth analysis of the human plasma proteome before and after RIPC. From the 68 protein markers reported in the literature, only 28 could be mapped to manually reviewed (Swiss-Prot) protein sequences. 23 of them were monitored in our untargeted experiment. However, their significant regulation could not be reproducibly estimated. In fact, among the 394 plasma proteins we accurately quantified, no significant regulation could be confidently and reproducibly assessed. This indicates that it is difficult to both monitor and reproduce published data from experiments exploring for RIPC induced plasma proteomic regulations, and suggests that further work should be directed towards small humoral factors. To simplify this task, we made our proteomic dataset available via ProteomeXchange, where scientists can mine for novel potential targets.     

Tissue MicroRNAs as Predictors of Outcome in Patients with Metastatic Colorectal Cancer Treated with First Line Capecitabine and Oxaliplatin with or without Bevacizumab

Purpose

We tested the hypothesis that expression of microRNAs (miRNAs) in cancer tissue can predict effectiveness of bevacizumab added to capecitabine and oxaliplatin (CAPEOX) in patients with metastatic colorectal cancer (mCRC).

Experimental Design

Patients with mCRC treated with first line CAPEOX and bevacizumab (CAPEOXBEV): screening (n = 212) and validation (n = 121) cohorts, or CAPEOX alone: control cohort (n = 127), were identified retrospectively and archival primary tumor samples were collected. Expression of 754 miRNAs was analyzed in the screening cohort using polymerase chain reaction (PCR) arrays and expression levels were related to time to disease progression (TTP) and overall survival (OS). Significant miRNAs from the screening study were analyzed in all three cohorts using custom PCR arrays. In situ hybridization (ISH) was done for selected miRNAs.

Results

In the screening study, 26 miRNAs were significantly correlated with outcome in multivariate analyses. Twenty-two miRNAs were selected for further study. Higher miR-664-3p expression and lower miR-455-5p expression were predictive of improved outcome in the CAPEOXBEV cohorts and showed a significant interaction with bevacizumab effectiveness. The effects were strongest for OS. Both miRNAs showed high expression in stromal cells. Higher expression of miR-196b-5p and miR-592 predicted improved outcome regardless of bevacizumab treatment, with similar effect estimates in all three cohorts.

Conclusions

We have identified potentially predictive miRNAs for bevacizumab effectiveness and additional miRNAs that could be related to chemotherapy effectiveness or prognosis in patients with mCRC. Our findings need further validation in large cohorts, preferably from completed randomized trials.     

Modelling the Time Evolution of Active Caspase-3 Protein in the Rat Lens after In Vivo Exposure to Ultraviolet Radiation-B

Purpose

To introduce a model for the time evolution of active caspase-3 protein expression in albino rat lens up to 24 hours after in vivo exposure to low dose UVR in the 300 nm wavelength region (UVR-300 nm).

Methods

Forty Sprague-Dawley rats were unilaterally exposed in vivo to 1 kJ/m² UVR-300 nm for 15 minutes. At 0.5, 8, 16, and 24 hours after the UVR exposure, the exposed and contralateral not-exposed lenses were removed and processed for immunohistochemistry. The differences in the probability of active caspase-3 expression at four different time points after exposure were used to determine the time evolution of active caspase-3 expression. A logistic model was introduced for the expression of active caspase-3. The parameters for the exposed and the not exposed lenses were estimated for the observation time points.

Results

The exposure to UVR-300 nm impacted on the parameters of the logistic model. Further, the parameters of the model varied with time after exposure to UVR-300 nm.

Conclusion

The logistic model predicts the impact of exposure to UVR-300 nm on the spatial distribution of probability of active caspase-3 protein expression, depending on time.