Alphavirus Replicon Particles Expressing TRP-2 Provide Potent Therapeutic Effect on Melanoma through Activation of Humoral and Cellular Immunity

Background

Malignant melanoma is the deadliest form of skin cancer and is refractory to conventional chemotherapy and radiotherapy. Therefore alternative approaches to treat this disease, such as immunotherapy, are needed. Melanoma vaccine design has mainly focused on targeting CD8⁺ T cells. Activation of effector CD8⁺ T cells has been achieved in patients, but provided limited clinical benefit, due to immune-escape mechanisms established by advanced tumors. We have previously shown that alphavirus-based virus-like replicon particles (VRP) simultaneously activate strong cellular and humoral immunity against the weakly immunogenic melanoma differentiation antigen (MDA) tyrosinase. Here we further investigate the antitumor effect and the immune mechanisms of VRP encoding different MDAs.

Methodology/Principal Findings

VRP encoding different MDAs were screened for their ability to prevent the growth of the B16 mouse transplantable melanoma. The immunologic mechanisms of efficacy were investigated for the most effective vaccine identified, focusing on CD8⁺ T cells and humoral responses. To this end, ex vivo immune assays and transgenic mice lacking specific immune effector functions were used. The studies identified a potent therapeutic VRP vaccine, encoding tyrosinase related protein 2 (TRP-2), which provided a durable anti-tumor effect. The efficacy of VRP-TRP2 relies on a novel immune mechanism of action requiring the activation of both IgG and CD8⁺ T cell effector responses, and depends on signaling through activating Fcγ receptors.

Conclusions/Significance

This study identifies a VRP-based vaccine able to elicit humoral immunity against TRP-2, which plays a role in melanoma immunotherapy and synergizes with tumor-specific CD8⁺ T cell responses. These findings will aid in the rational design of future immunotherapy clinical trials.     

Quality assessed nonculture techniques for detection and typing of meningococci

PCR protocols are increasingly used in laboratories worldwide for the diagnosis and confirmation of invasive meningococcal infection. Protocols are now available for the identification of Neisseria meningitidis, for genogrouping, susceptibility to antibiotics and genotyping of the corresponding isolates. The implementation of quality assurance (QA) schemes and standardization of protocols are required. Diagnostic and confirmatory PCRs should perform consistently in clinical and reference microbiology laboratories. General QA schemes address the issues of sample preparation, PCR laboratory environment, equipment and validation of protocols. Moreover, external QA interlaboratory studies are essential. The European Monitoring Group on Meningococci has provided a good forum to conduct such studies through the development and distribution of samples and protocols for nonculture detection and typing of N. meningitidis.     

Increase in Fe2+-producing activity during growth of Acidithiobacillus ferrooxidans ATCC23270 on sulfur

When Acidithiobacillus ferrooxidans ATCC23270 cells, grown for many generations on sulfur were grown in sulfur medium with and without Fe(3+), the bacterium markedly increased not only in iron oxidase activity but also in Fe(2+)-producing sulfide:ferric ion oxidoreductase (SFORase) activity during the early log phase, and retained part of these activities during the late log phase. The activity of SFORase, which catalyzes the production of Fe(2+) from Fe(3+) and sulfur, of sulfur-grown cells was approximately 10-20 fold higher than that of iron-grown cells. aa(3) type cytochrome c oxidase, an important component of iron oxidase in A. ferrooxidans, was partially purified from sulfur-grown cells. A. ferrooxidans ATCC23270 cells grown for many generations on sulfur had the ability to grow on iron as rapidly as that did iron-grown cells. These results suggest that both iron oxidase and Fe(2+)-producing SFORase have a role in the energy generation of A. ferrooxidans ATCC23270 from sulfur.     

Impact of nano-zinc-oxide as an alternative source of zinc in date palm culture media

Plant Cell, Tissue and Organ Culture (PCTOC) - Leaf chlorosis is often a problem in micropropagated Rubus idaeus, which makes successful acclimatization difficult. We found that...     

Experimental and probabilistic analysis of distal femoral periprosthetic fracture: a comparison of locking plate and intramedullary nail fixation. Part B: probabilistic investigation

The following is Part B of a two-part study. Part A evaluated, biomechanically, intramedullary (IM) nails versus locking plates for fixation of an extra-articular, metaphyseal wedge fracture in synthetic osteoporotic bone. Part B of this study introduces deterministic finite element (FE) models of each construct type in synthetic osteoporotic bone and investigates the probability of periprosthetic fracture of the locking plate compared with the retrograde IM nail using Monte Carlo simulation. Deterministic FE models of the fractured femur implanted with IM nail and locking plate, respectively, were developed and validated using experimental data presented in Part A of this study. The models were validated by comparing the load-displacement curve of the experimental data with the load-displacement curve of the FE simulation with a root-mean square error of less than 3?mm. The validated FE models were then modified by defining the cortical and cancellous bone modulus of elasticity as uncertain variables that could be assumed to vary randomly. Monte Carlo simulation was used to evaluate the probability of fracture (POF) of each fixation. The POF represents the cumulative probability that the predicted shear stresses in the cortical bone will exceed the expected shear strength of the cortical bone. This investigation provides information regarding the significance of post-operative damage accumulation on the POF of the implanted bones when the two fixations are used. The probabilistic analysis found the locking plate fixation to have a higher POF than the IM nail fixation under the applied loading conditions (locking plate 21.8% versus IM nail 0.019%).     

Pre-ejection period by radial artery tonometry supplements echo doppler findings during biventricular pacemaker optimization

Background

Biventricular (Biv) pacemaker echo optimization has been shown to improve cardiac output however is not routinely used due to its complexity. We investigated the role of a simple method involving computerized pre-ejection time (PEP) assessment by radial artery tonometry in guiding Biv pacemaker optimization.

Methods

Blinded echo and radial artery tonometry were performed simultaneously in 37 patients, age 69.1 ± 12.8 years, left ventricular (LV) ejection fraction (EF) 33 ± 10%, during Biv pacemaker optimization. Effect of optimization on echo derived velocity time integral (VTI), ejection time (ET), myocardial performance index (MPI), radial artery tonometry derived PEP and echo-radial artery tonometry derived PEP/VTI and PEP/ET indices was evaluated.

Results

Significant improvement post optimization was achieved in LV ET (286.9 ± 37.3 to 299 ± 34.6 ms, p < 0.001), LV VTI (15.9 ± 4.8 cm to 18.4 ± 5.1 cm, p < 0.001) and MPI (0.57 ± 0.2 to 0.45 ± 0.13, p < 0.001) and in PEP (246.7 ± 36.1 ms to 234.7 ± 35.5 ms, p = 0.003), PEP/ET (0.88 ± 0.21 to 0.79 ± 0.17, p < 0.001), and PEP/VTI (17.3 ± 7 to 13.78 ± 4.7, p < 0.001). The correlation between comprehensive echo Doppler and radial artery tonometry-PEP guided optimal atrioventricular delay (AVD) and optimal interventricular delay (VVD) was 0.75 (p < 0.001) and 0.69 (p < 0.001) respectively. In 29 patients with follow up assessment, New York Heart Association (NYHA) class reduced from 2.5 ± 0.8 to 2.0 ± 0.9 (p = 0.004) at 1.8 ± 1.4 months.

Conclusion

An acute shortening of PEP by radial artery tonometry occurs post Biv pacemaker optimization and correlates with improvement in hemodynamics by echo Doppler and may provide a cost-efficient approach to assist with Biv pacemaker echo optimization.     

Evaluation of absolute peptide quantitation strategies using selected reaction monitoring

The use of internal peptide standards in selected reaction monitoring experiments enables absolute quantitation. Here, we describe three approaches addressing calibration of peptide concentrations in complex matrices and assess their performance in terms of trueness and precision. The simplest approach described is single reference point quantitation where a heavy peptide is spiked into test samples and the endogenous analyte quantified relative to the heavy peptide internal standard. We refer to the second approach as normal curve quantitation. Here, a constant amount of heavy peptide and a varying amount of light peptide are spiked into matrix to construct a calibration curve. This accounts for matrix effects but due to the presence of endogenous analyte, it is usually not possible to determine the lower LOQ. We refer to the third method as reverse curve quantitation. Here, a constant amount of light peptide and a varying amount of heavy peptide are spiked into matrix to construct a calibration curve. Because there is no contribution to the heavy peptide signal from endogenous analyte, it is possible to measure the equivalent of a blank sample and determine LOQ. These approaches are applied to human plasma samples and used to assay peptides of a set of apolipoproteins.     

Absent or delayed adrenarche in Pit-1/POU1F1 deficiency

Mutations of the PIT1/POU1F1 gene are responsible for a rare variant of anterior hypopituitarism, including deficiency of growth hormone, prolactin and thyrotropin. In 8 ethnically diverse POU1F1-deficient patients (4 different mutations) with normal circulating levels of cortisol and adrenocorticotropic hormone, and with spontaneous onset and progression of puberty, we observed an absence or delay of adrenarche (median circulating dehydroepiandrosterone-sulfate -6.2 SD); in each of the 4 postmenarcheal females, pubarche (i.e. appearance of pubic hair) was also absent or delayed. The absence/delay of adrenarche in POU1F1-deficient patients and the absence/delay of pubarche in POU1F1-deficient females suggest that a POU1F1-dependent factor contributes to the normal development of adrenarche and female pubarche.     

Amelioration of altered antioxidant status and membrane linked functions by vanadium andTrigonella in alloxan diabetic rat brains

Trigonella foenum graecum seed powder (TSP) and sodium orthovanadate (SOV) have been reported to have antidiabetic effects. However, SOV exerts hypoglycemic effects at relatively high doses with several toxic effects. We used low doses of vanadate in combination with TSP and evaluated their antidiabetic effects on antioxidant enzymes and membrane-linked functions in diabetic rat brains. In rats, diabetes was induced by alloxan monohydrate (15 mg/100 g body wt.) and they were treated with 2 IU insulin, 0.6 mg/ml SOV, 5% TSP and a combination of 0.2 mg/ml SOV with 5% TSP for 21 days. Blood glucose levels, activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), Na⁺/K⁺ ATPase, membrane lipid peroxidation and fluidity were determined in different fractions of whole brain after 21 days of treatment. Diabetic rats showed high blood glucose (P < 0.001), decreased activities of SOD, catalase and Na⁺/K⁺ ATPase (P < 0.01,P < 0.001 andP < 0.01), increased levels of GPx and MDA (P < 0.01 andP < 0.001) and decreased membrane fluidity (P < 0.01). Treatment with different antidiabetic compounds restored the above-altered parameters. Combined dose ofTrigonella and vanadate was found to be the most effective treatment in normalizing these alterations. Lower doses of vanadate could be used in combination with TSP to effectively counter diabetic alterations without any toxic effects.