Multimarker scores of Th1 and Th2 immune cellular profiles in peripheral blood predict response and immune related toxicity with CTLA4 blockade and IFNα in melanoma

Neoadjuvant therapy with ipilimumab in combination with high dose IFNα was evaluated in patients with locally/regionally advanced melanoma in a previously reported clinical trial [NCT01608594]. In this study, peripheral immune cell profiling was performed in order to investigate the underlying mechanisms of tumor immune susceptibility and resistance. Peripheral blood mononuclear cells (PBMCs) from treated patients (N = 28) were collected at baseline and then at 6-weeks, 3-months and 12-months. High complexity (14-color) flow cytometry, designed to detect key immunological biomarkers was used to evaluate the frequencies of immune cell subsets. Statistical significance was determined using R-package employing Kruskal's test. We found that higher levels of Th1 cells at baseline (defined as CD45RA- CCR6- CXCR3+ CCR4-) correlated with the preoperative radiological response (p = 0.007) while higher Th2 cells (defined as CD45RA- CCR6- CXCR3- CCR4+) were associated with progressive disease (p = 0.009). A multimarker score consisting of higher levels of Th1 cells and CD8+ central memory T-cells was associated with pathologic complete response (pCR) (p = 0.041) at surgical resection. On the other hand, high TIM3 expression on T-cells correlated with gross viable tumor (p = 0.047). With regard to immune related toxicity, higher levels of phenotypically naive (defined as CCR7+CD45RA+) and effector memory (defined as CCR7-CD45RO+) CD8+ T-cells (p = 0.014) or lower levels of Th2 cells were associated with lower toxicity (p = 0.024). Furthermore, a multimarker score consisting of higher CD19+ and CD8+ cells was associated with lower toxicity (p = 0.0014). In conclusion, our study yielded mechanistic insights related to the immune impact of CTLA4 blockade and IFNα and potential biomarkers of immune response and toxicity.     

BDC12-4.1 T-Cell Receptor Transgenic Insulin-Specific CD4 T Cells Are Resistant to In Vitro Differentiation into Functional Foxp3+ T Regulatory Cells

The infusion of ex vivo-expanded autologous T regulatory (Treg) cells is potentially an effective immunotherapeutic strategy against graft-versus-host disease (GvHD) and several autoimmune diseases, such as type 1 diabetes (T1D). However, in vitro differentiation of antigen-specific T cells into functional and stable Treg (iTreg) cells has proved challenging. As insulin is the major autoantigen leading to T1D, we tested the capacity of insulin-specific T-cell receptor (TCR) transgenic CD4⁺ T cells of the BDC12-4.1 clone to convert into Foxp3⁺ iTreg cells. We found that in vitro polarization toward Foxp3⁺ iTreg was effective with a majority (>70%) of expanded cells expressing Foxp3. However, adoptive transfer of Foxp3⁺ BDC12-4.1 cells did not prevent diabetes onset in immunocompetent NOD mice. Thus, in vitro polarization of insulin-specific BDC12-4.1 TCR transgenic CD4⁺ T cells toward Foxp3⁺ cells did not provide dominant tolerance in recipient mice. These results highlight the disconnect between an in vitro acquired Foxp3⁺ cell phenotype and its associated in vivo regulatory potential.     

Kinetics of hydrolysis of acetyl, valeroyl and nicotinoyl acyl derivatives of stobadine

The present work deals with the kinetics of hydrolysis of the acyl derivatives of stobadine, an originally synthesized potential antiarrhythmic and antihypoxic drug, which was found to have also an excellent scavenging effect on reactive oxygen species. The acyl derivatives of stobadine, which possess high lipophilicity, represent model blood-brain barrier penetrating agents. It is assumed that the acyl derivatives of stobadine may act as prodrugs which are hydrolysed in different biological tissues to release the active drug. The decomposition of three acyl derivatives of stobadine was studied in acidic, basic and neutral buffer solutions at constant ionic strength (0.1 mol/L) at 25 degrees and 70 degrees C using UV spectrophotometric method. The pseudo first-order rate constants and the pH-rate profile for the degradation of acetyl-, valeroyl- and nicotinoyl-derivatives of stobadine were determined. Confirmation that stobadine was the first degradation product was provided by thin-layer chromatography.     

Temporal Intra-Individual Variation of Immunological Biomarkers in Type 1 Diabetes Patients: Implications for Future Use in Cross-Sectional Assessment

Multiple immune parameters such as frequencies of autoreactive CD4⁺, CD8⁺ T-cells and CD4⁺CD25⁺Foxp3⁺ T-cells have been explored as biomarkers in human T1D. However, intra-individual temporal variation of these parameters has not been assessed systematically over time. We determined the variation in each of these parameters in a cohort of T1D and healthy donors (HDs), at monthly intervals for one year. Despite low intra- and inter-assay co-efficient of variation (CV), mean CVs for each of the immune parameters were 119.1% for CD4⁺ T-cell-derived IFN-γ, 50.44% for autoreactive CD8⁺ T-cells, and 31.24% for CD4⁺CD25⁺Foxp3⁺ T-cells. Further, both HDs and T1D donors had similar CVs. The variation neither correlated with BMI, age, disease duration or insulin usage, nor were there detectable cyclical patterns of variation. However, averaging results from multiple visits for an individual provided a better estimate of the CV between visits. Based on our data we predict that by averaging values from three visits a treatment effect on these parameters with a 50% effect size could be detected with the same power using 1.8–4-fold fewer patients within a trial compared to using values from a single visit. Thus, our present data contribute to a more robust, accurate endpoint design for future clinical trials in T1D and aid in the identification of truly efficacious therapies.     

Indirect stimulation of human Vγ2Vδ2 T cells through alterations in isoprenoid metabolism

Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the 2-C-methyl-d-erythritol-4-phosphate pathway used by microbes, and isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway used by humans. Aminobisphosphonates and alkylamines indirectly stimulate Vγ2Vδ2 cells by inhibiting farnesyl diphosphate synthase (FDPS) in the mevalonate pathway, thereby increasing IPP/triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester that directly stimulate. In this study, we further characterize stimulation by these compounds and define pathways used by new classes of compounds. Consistent with FDPS inhibition, stimulation of Vγ2Vδ2 cells by aminobisphosphonates and alkylamines was much more sensitive to statin inhibition than stimulation by prenyl pyrophosphates; however, the continuous presence of aminobisphosphonates was toxic for T cells and blocked their proliferation. Aminobisphosphonate stimulation was rapid and prolonged, independent of known Ag-presenting molecules, and resistant to fixation. New classes of stimulatory compounds-mevalonate, the alcohol of HMBPP, and alkenyl phosphonates-likely stimulate differently. Mevalonate, a rate-limiting metabolite, appears to enter cells to increase IPP levels, whereas the alcohol of HMBPP and alkenyl phosphonates are directly recognized. The critical chemical feature of bisphosphonates is the amino moiety, because its loss switched aminobisphosphonates to direct Ags. Transfection of APCs with small interfering RNA downregulating FDPS rendered them stimulatory for Vγ2Vδ2 cells and increased cellular IPP. Small interfering RNAs for isopentenyl diphosphate isomerase functioned similarly. Our results show that a variety of manipulations affecting isoprenoid metabolism lead to stimulation of Vγ2Vδ2 T cells and that pulsing aminobisphosphonates would be more effective for the ex vivo expansion of Vγ2Vδ2 T cells for adoptive cancer immunotherapy.     

Apoptotic and antiapoptotic activity of L protein of Theiler's murine encephalomyelitis virus

Cellular apoptosis induced by viral genes can play a critical role in determining virulence as well as viral persistence. This form of cell death has been of interest with respect to Theiler's murine encephalomyelitis virus (TMEV) because the GDVII strain and members of the GDVII subgroup are highly neurovirulent, while the DA strain and members of the TO subgroup induce a chronic progressive inflammatory demyelination with persistence of the virus in the central nervous system. The TMEV L protein has been identified as important in the pathogenesis of Theiler's virus-induced demyelinating disease (TMEV-IDD). We now show that DA L is apoptotic following transfection of L expression constructs or following DA virus infection of HeLa cells; the apoptotic activity depends on the presence of the serine/threonine domain of L, especially a serine at amino acid 57. In contrast, GDVII L has little apoptotic activity following transfection of L expression constructs in HeLa cells and is antiapoptotic following GDVII infection of HeLa cells. Of note, both DA and GDVII L cleave caspase-3 in BHK-21 cells, although neither implements the full apoptotic machinery in this cell type as manifested by the induction of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. The differences in apoptotic activities of DA and GDVII L in varied cell types may play an important role in TMEV subgroup-specific disease phenotypes.     

The duality of fractals: roughness and self-similarity

The fractal dimension (D _HB) is an interesting metrics because it is supposed to quantify by a single value, scale independence and roughness of ecological objects. However, we show here that those two properties may be quantified by a single dimension only in some specific cases. In general, a non-integer D _HB quantifies only the roughness, and self-similarity needs to be evidenced or postulated by other means. Second, we revisit some aspects of the practical estimation of D _HB. We recommend the use of madogram instead of variogram for estimations based on geostatistics. We propose a simplification of its estimation for 2D fields and discuss its possible relationship with self-similarity. We finally underline the problem of scale and resolution. Field data recorded during a scientific acoustic survey on the North Sea herring are used for illustrations. The paper concludes on a synthesis of practical recommendations to ecologists when using fractal dimension.     

Transformation maligne d’un goitre ovarien. À propos d’une observation

Struma ovarii is a monodermal variant of ovarian teratoma, which predominantly contains thyroid tissue. Malignant transformation and metastasis are very rare. The treatment of malignant struma ovarii remains controversial. We report the case of a patient with a recurrent struma ovarii, treated 18 years ago. The malignant transformation and metastases were treated with a combination of surgery and I¹³¹ ablation therapy.