TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2.

Tumor necrosis factor (TNF)-α is a pleiotropic cytokine implicated in the etiology of several autoimmune diseases, including rheumatoid arthritis (RA). TNF-α regulates diverse effector functions through the activation of TNF-α receptor (TNFR)1 and TNFR2. Although the detrimental role of this cytokine has been addressed in distinct disease settings, the effects of TNF-α on cytokine production by isolated CD4+ T helper type 1 (Th1) and Th17 cells, two T cell subpopulations that contribute to the pathogenesis of RA, have not been completely elucidated. Here, we show that TNF-α promotes a reduction and expansion in the frequency of both T cell subsets producing IFN-γ and IL-17, respectively. Selective blockade of TNFR1 or TNFR2 on Th1 and Th17 cells revealed that TNFR2 mediates the decrease in IFN-γ production, while signaling through both receptors augments IL-17 production. We also demonstrate that Th1, but not Th17 cells from RA patients present lower levels of TNFR1 compared to healthy controls, whereas TNFR2 expression on both T cell types is similar between patients and controls. Since TNF-α receptors levels in RA patients are not significantly changed by the therapeutic blockade of TNF-α, we propose that targeting TNFR2 may represent an alternative strategy to normalize the levels of key cytokines that contribute to RA pathogenesis.

In vitro Treatment of a Murine Mammary Adenocarcinoma Cell Line with Recombinant Trypanosoma cruzi Calreticulin Promotes Immunogenicity and Phagocytosis.

American Trypanosomiasis, a parasitic disease produced by Trypanosoma cruzi (T. cruzi), endemic in Latin America, infects about 6 million people. During the chronic stage of the infection, approximately 30% of infected people will develop Chagas Disease, the clinical manifestation. Few decades ago it was reported that, during the chronic stage, the parasite interferes with the development of solid tumors. However, the identification of parasite molecules responsible for such effects remained elusive. Years later, we described T.cruzi Calreticulin (TcCalr), an endoplasmic reticulum resident chaperone that infective trypomastigotes translocate to the parasite exterior, where it displays anticomplement activities. Most likely, at least some of these activities are related with the antitumor properties of TcCalr, as shown in in vitro, ex vivo, in ovum, and in vivo models. In this context we, we have seen that in vivo subcutaneous peritumoral inoculation of rTcCalr enhances local infiltration of T cells and slows tumor development. Based on these precedents, we propose that in vitro treatment of a mammary adenocarcinoma (TA3 cell line) with rTcCalr, will enhance tumor immunogenicity. In agreement with this proposal, we have shown that: i). rTcCalr binds to TA3 cells in a concentration-dependent fashion, ii). C1q binds to TA3 cells in an rTcCalr-dependent fashion, confirmed by the reversion attained using anti-TcS (a central TcCalr domain that binds C1) F(ab')2 antibody fragments, iii). incubation of TA3 cells with rTcCalr, promotes cell phagocytosis by murine macrophages and, iv). rTcCalr decreases the membrane expression of MHC class II, m-Dectin-1, Galectin-9 and PD-L1, while increasing the expression of Rae-1γ. In synthesis, herein we show that in vitro treatment of a murine mammary adenocarcinoma with rTcCalr enhances phagocytosis and modulates the expression of a variety of membrane molecules that correlates with increased tumor immunogenicity.

Decreased invariant natural killer T-cell-mediated antitumor immune response in patients with gastric cancer.

Gastric cancer (GC) is the third most common cause of cancer-related death worldwide. Invariant natural killer T (iNKT) cells are innate-like cytotoxic T lymphocytes involved in tumor immune surveillance. They can be activated either through CD1d-presented glycolipid antigens recognized by their invariant T-cell receptor, cytokines or by sensing tumor-associated stress-induced ligands through the natural killer group 2, member D (NKG2D) receptor. Although the number and functionality of iNKT cells may be decreased in several types of cancer, here we show that GC patients presented a mild increase in iNKT cell frequencies and numbers in the blood compared with healthy donors. In GC patients, iNKT cells, expanded in vitro with α-galactosyl ceramide and stimulated with phorbol 12-myristate 13-acetate and ionomycin, produced higher levels of interleukin-2 and transforming growth factor-beta, while their capacity to degranulate remained preserved. Because tumor-derived epithelial cell adhesion molecule-positive epithelial cells did not display surface CD1d, and NKG2D ligands (NKG2DLs) were detected in the gastric tumor milieu, we envisioned a role for NKG2D in iNKT cell functions. Peripheral iNKT cells from GC patients and controls presented similar levels of NKG2D; nevertheless, the percentages of interferon-γ-producing and CD107a-positive iNKT cells from patients were reduced upon challenge with CD1d-negative, NKG2DL-positive K562 cells, suggesting a compromised response by iNKT cells in GC patients, which may not result from impaired NKG2D/NKG2DL signaling. The decreased response of iNKT cells may explain the fact that higher frequencies of circulating iNKT cells did not confer a survival benefit for GC patients. Therefore, functional impairment of iNKT cells in GC may contribute to tumor immune escape and favor disease progression.

Regulation of Tolerogenic Features on Dexamethasone-Modulated MPLA-Activated Dendritic Cells by MYC.

The potential of tolerogenic dendritic cells (tolDCs) to shape immune responses and restore tolerance has turn them into a promising therapeutic tool for cellular therapies directed toward immune regulation in autoimmunity. Although the cellular mechanisms by which these cells can exert their regulatory function are well-known, the mechanisms driving their differentiation and function are still poorly known, and the variety of stimuli and protocols applied to differentiate DCs toward a tolerogenic phenotype makes it even more complex to underpin the molecular features involved in their function. Through transcriptional profiling analysis of monocyte-derived tolDCs modulated with dexamethasone (Dex) and activated with monophosphoryl lipid A (MPLA), known as DM-DCs, we were able to identify MYC as one of the transcriptional regulators of several genes differentially expressed on DM-DCs compared to MPLA-matured DCs (M-DCs) and untreated/immature DCs (DCs) as revealed by Ingenuity Pathway Analysis (IPA) upstream regulators evaluation. Additionally, MYC was also amidst the most upregulated genes in DM-DCs, finding that was confirmed at a transcriptional as well as at a protein level. Blockade of transactivation of MYC target genes led to the downregulation of tolerance-related markers IDO1 and JAG1. MYC blockade also led to downregulation of PLZF and STAT3, transcription factors associated with immune regulation and inhibition of DC maturation, further supporting a role of MYC as an upstream regulator contributing to the regulatory phenotype of DM-DCs. On the other hand, we had previously shown that fatty acid oxidation, oxidative metabolism and zinc homeostasis are amongst the main biological functions represented in DM-DCs, and here we show that DM-DCs exhibit higher intracellular expression of ROS and Zinc compared to mature M-DCs and DCs. Taken together, these findings suggest that the regulatory profile of DM-DCs is partly shaped by the effect of the transcriptional regulation of tolerance-inducing genes by MYC and the modulation of oxidative metabolic processes and signaling mediators such as Zinc and ROS.

Mammographic density: Comparison of visual assessment with fully automatic calculation on a multivendor dataset.

OBJECTIVES: To compare breast density (BD) assessment provided by an automated BD evaluator (ABDE) with that provided by a panel of experienced breast radiologists, on a multivendor dataset. METHODS: Twenty-one radiologists assessed 613 screening/diagnostic digital mammograms from nine centers and six different vendors, using the BI-RADS a, b, c, and d density classification. The same mammograms were also evaluated by an ABDE providing the ratio between fibroglandular and total breast area on a continuous scale and, automatically, the BI-RADS score. A panel majority report (PMR) was used as reference standard. Agreement (κ) and accuracy (proportion of cases correctly classified) were calculated for binary (BI-RADS a-b versus c-d) and 4-class classification. RESULTS: While the agreement of individual radiologists with the PMR ranged from κ = 0.483 to κ = 0.885, the ABDE correctly classified 563/613 mammograms (92 %). A substantial agreement for binary classification was found for individual reader pairs (κ = 0.620, standard deviation [SD] = 0.140), individual versus PMR (κ = 0.736, SD = 0.117), and individual versus ABDE (κ = 0.674, SD = 0.095). Agreement between ABDE and PMR was almost perfect (κ = 0.831). CONCLUSIONS: The ABDE showed an almost perfect agreement with a 21-radiologist panel in binary BD classification on a multivendor dataset, earning a chance as a reproducible alternative to visual evaluation. KEY POINTS: Individual BD assessment differs from PMR with κ as low as 0.483. An ABDE correctly classified 92 % of mammograms with almost perfect agreement (κ = 0.831). An ABDE can be a valid alternative to subjective BD assessment.

Novel Gallate Triphenylphosphonium Derivatives with Potent Antichagasic Activity.

disease is one of the most neglected tropical diseases in the world, affecting nearly 15 million people, primarily in Latin America. Only two drugs are used for the treatment of this disease, nifurtimox and benznidazole. These drugs have limited efficacy and frequently induce adverse effects, limiting their usefulness. Consequently, new drugs must be found. In this study, we demonstrated the in vitro trypanocidal effects of a series of four gallic acid derivatives characterized by a gallate group linked to a triphenylphosphonium (TPP(+)) moiety (a delocalized cation) via a hydrocarbon chain of 8, 10, 11, or 12 atoms (TPP(+)-C8, TPP(+)-C10, TPP(+)-C11, and TPP(+)-C12, respectively). We analyzed parasite viability in isolated parasites (by MTT reduction and flow cytometry) and infected mammalian cells using T. cruzi Y strain trypomastigotes. Among the four derivatives, TPP(+)-C10 and TPP(+)-C12 were the most potent in both models, with EC50 values (in isolated parasites) of 1.0 ± 0.6 and 1.0 ± 0.7 µM, respectively, and were significantly more potent than nifurtimox (EC50 = 4.1 ± 0.6 µM). At 1 µM, TPP(+)-C10 and TPP(+)-C12 induced markers of cell death, such as phosphatidylserine exposure and propidium iodide permeabilization. In addition, at 1 µM, TPP(+)-C10 and TPP(+)-C12 significantly decreased the number of intracellular amastigotes (TPP(+)-C10: 24.3%, TPP(+)-C12: 19.0% of control measurements, as measured by DAPI staining) and the parasite's DNA load (C10: 10%, C12: 13% of control measurements, as measured by qPCR). Based on the previous mode of action described for these compounds in cancer cells, we explored their mitochondrial effects in isolated trypomastigotes. TPP(+)-C10 and TPP(+)-C12 were the most potent compounds, significantly altering mitochondrial membrane potential at 1 µM (measured by JC-1 fluorescence) and inducing mitochondrial transition pore opening at 5 µM. Taken together, these results indicate that the TPP(+)-C10 and TPP(+)-C12 derivatives of gallic acid are promising trypanocidal agents with mitochondrial activity.

Breast Cancer: Computer-aided Detection with Digital Breast Tomosynthesis.

PURPOSE: To evaluate a commercial tomosynthesis computer-aided detection (CAD) system in an independent, multicenter dataset. MATERIALS AND METHODS: Diagnostic and screening tomosynthesis mammographic examinations (n = 175; cranial caudal and mediolateral oblique) were randomly selected from a previous institutional review board-approved trial. All subjects gave informed consent. Examinations were performed in three centers and included 123 patients, with 132 biopsy-proven screening-detected cancers, and 52 examinations with negative results at 1-year follow-up. One hundred eleven lesions were masses and/or microcalcifications (72 masses, 22 microcalcifications, 17 masses with microcalcifications) and 21 were architectural distortions. Lesions were annotated by radiologists who were aware of all available reports. CAD performance was assessed as per-lesion sensitivity and false-positive results per volume in patients with negative results. RESULTS: Use of the CAD system showed per-lesion sensitivity of 89% (99 of 111; 95% confidence interval: 81%, 94%), with 2.7 ± 1.8 false-positive rate per view, 62 of 72 lesions detected were masses, 20 of 22 were microcalcification clusters, and 17 of 17 were masses with microcalcifications. Overall, 37 of 39 microcalcification clusters (95% sensitivity, 95% confidence interval: 81%, 99%) and 79 of 89 masses (89% sensitivity, 95% confidence interval: 80%, 94%) were detected with the CAD system. On average, 0.5 false-positive rate per view were microcalcification clusters, 2.1 were masses, and 0.1 were masses and microcalcifications. CONCLUSION: A digital breast tomosynthesis CAD system can allow detection of a large percentage (89%, 99 of 111) of breast cancers manifesting as masses and microcalcification clusters, with an acceptable false-positive rate (2.7 per breast view). Further studies with larger datasets acquired with equipment from multiple vendors are needed to replicate the findings and to study the interaction of radiologists and CAD systems.

Tolerogenic dendritic cells derived from donors with natural rubber latex allergy modulate allergen-specific T-cell responses and IgE production.

Natural rubber latex (NRL; Hevea brasiliensis) allergy is an IgE-mediated reaction to latex proteins. When latex glove exposure is the main sensitizing agent, Hev b 5 is one of the major allergens. Dendritic cells (DC), the main antigen presenting cells, modulated with pharmacological agents can restore tolerance in several experimental models, including allergy. In the current study, we aimed to generate DC with tolerogenic properties from NRL-allergic patients and evaluate their ability to modulate allergen-specific T and B cell responses. Here we show that dexamethasone-treated DC (dxDC) differentiated into a subset of DC, characterized by low expression of MHC class II, CD40, CD80, CD86 and CD83 molecules. Compared with LPS-matured DC, dxDC secreted lower IL-12 and higher IL-10 after CD40L activation, and induced lower alloantigenic T cell proliferation. We also show that dxDC pulsed with the dominant Hev b 5 T-cell epitope peptide, Hev b 5(46-65), inhibited both proliferation of Hev b 5-specific T-cell lines and the production of Hev b 5-specific IgE. Additionally, dxDC induced a subpopulation of IL-10-producing regulatory T cells that suppressed proliferation of Hev b 5-primed T cells. In conclusion, dxDC generated from NRL-allergic patients can modulate allergen-specific T-cell responses and IgE production, supporting their potential use in allergen-specific immunotherapy.

A first evaluation of breast radiological density assessment by QUANTRA software as compared to visual classification.

Breast radiological density is a determinant of breast cancer risk and of mammography sensitivity and may be used to personalize screening approach. We first analyzed the reproducibility of visual density assessment by eleven experienced radiologists classifying a set of 418 digital mammograms: reproducibility was satisfactory on a four (BI-RADS D1-2-3-4: weighted kappa = 0.694-0.844) and on a two grade (D1-2 vs D3-4: kappa = 0.620-0.851), but subjects classified as with dense breast would range between 25.1 and 50.5% depending on the classifying reader. Breast density was then assessed by computer using the QUANTRA software which provided systematically lower density percentage values as compared to visual classification. In order to predict visual classification results in discriminating dense and non-dense breast subjects on a two grade scale (D3-4 vs, D1-2) the best fitting cut off value observed for QUANTRA was ≤22.0%, which correctly predicted 88.6% of D1-2, 89.8% of D3-4, and 89.0% of total cases. Computer assessed breast density is absolutely reproducible, and thus to be preferred to visual classification. Thus far few studies have addressed the issue of adjusting computer assessed density to reproduce visual classification, and more similar comparative studies are needed.

Anti-TNF therapy in patients with rheumatoid arthritis decreases Th1 and Th17 cell populations and expands IFN-γ-producing NK cell and regulatory T cell subsets.

The aim of this work was to study the effect of anti-TNF treatment on CD4+ Th1, Th17 and regulatory T cells (Tregs), together with CD8+ T cells and NK cells from rheumatoid arthritis (RA) patients. For this purpose, 18 RA patients received adalimumab during 16weeks and their peripheral blood lymphocytes were assessed by flow cytometry at the beginning and at the end of the study. We found that the proportion of Th17 cells was directly correlated with Th1 cells, but inversely correlated with IFN-γ-producing NK cells. A decrease was observed in Th1, Th17 cells and IFN-γ-producing CD8+ T cells by anti-TNF therapy. Conversely, the proportion of Tregs increased, as did the percentage of IFN-γ-producing NK cells. We postulate that a rise in IFN-γ production due to recovery of NK cells' function, together with expanded Tregs, contribute to decrease the Th17 response in anti-TNF-treated RA patients.

A synthetic peptide homologous to IL-10 functional domain induces monocyte differentiation to TGF-ß+ tolerogenic dendritic cells.

We have previously demonstrated that IT9302, a nonameric peptide homologous to the C-terminal domain of human IL-10, mimics several effects of the cytokine including down-regulation of the antigen presentation machinery and increased sensitivity of tumor cells to NK-mediated lysis. In the present report, we have explored a potential therapeutic utility for IT9302 related to the ex vivo production of tolerogenic dendritic cells (DCs). Our results indicate that IT9302 impedes human monocyte response to differentiation factors and reduces antigen presentation and co-stimulatory capacity by DCs. Additionally, peptide-treated DCs show impaired capacity to stimulate T-cell proliferation and IFN-γ production. IT9302 exerts its effect through mechanisms, in part, distinct from IL-10, involving STAT3 inactivation and NF-κB intracellular pathway. IT9302-treated DCs display increased expression of membrane-associated TGF-ß, linked to a more effective induction of foxp3+ regulatory T cells. These results illustrate for the first time that a short synthetic peptide can promote monocytes differentiation to tolerogenic DCs with therapeutic potential for the treatment of autoimmune and transplantation-related immunopathologic disease.

Inflammation, synovial angiogenesis and chondroid apoptosis in the evolution of type II collagen-induced arthritis.

Using the murine model of type II collagen-induced arthritis (CIA), we studied its evolution over time by histopathological, immunohistochemical and clinical evaluations. The first clinical symptoms appeared 28 days post-inoculation (dpi), with bovine type II collagen, with an average arthritic index of 1.00 +/- 0.48 corresponding to erythema of the articulation. The disease progressed, and by 70 dpi showed an average arthritic index of 3.83 +/- 0.27 corresponding to edema and maximum deformation, with ankylosis. Computed morphometry demonstrated that, in comparison to controls, the induction of CIA, produces a significant and increasing accumulation of inflammatory cells, fibrosis (p < 0.0001) and cartilage destruction (p = 0.0029). Likewise, the area of von Willebrand factor (vWF) immunostaining, as an indicator of endothelial proliferation, increased significantly from 28 dpi (p < 0.0001), in CIA mice compared to controls. However, the effective synovial vascularization, calculated as the synovial vascular bed area index, significantly increased by 42 dpi (p = 0.0014). This indicates that the activation and proliferation of endothelium becomes significant before an effective vascularization area is formed. The apoptosis index was also an earlier indicator of cartilage damage, becoming significant from 28 dpi in comparison to controls (p < 0.0001). Finally, it was observed that the increase in the arthritic index showed a strong correlation with the increase in both angiogenesis (r = 0.95; p = 0.0021) and apoptosis (r = 0.90; p = 0.0015). In conclusion, a robust correlation between synovial membrane inflammation, angiogenesis and chondrocyte apoptosis, with respect to the increase in the clinical severity of CIA, has been demonstrated by a quantitative computer-assisted immunomorphometric analysis.

Short-term lipopolysaccharide stimulation induces differentiation of murine bone marrow-derived dendritic cells into a tolerogenic phenotype.

Dendritic cells (DCs) are professional, antigen-presenting cells, which induce and regulate T cell reactivity. DCs are crucial in innate and adaptive immune responses, and are also involved in central and peripheral tolerance induction. Tolerance can be mediated by immature and semi-mature DCs expressing low levels of co-stimulator and major histocompatibility complex (MHC) molecules. The aim of this study was to investigate the ability of short-term lipopolysaccharide (LPS) stimulation to modulate the stage of differentiation of bone marrow-derived DCs. For this purpose, DCs obtained from DBA1/lacJ mice were stimulated for four (4hLPS/DCs) or 24 (24hLPS/DCs) hours with LPS, using DCs without stimulation (0hLPS/DCs) as a control. Flow cytometry analysis of 4hLPS/DCs showed intermediate CD40 and MHC class II expression, lower than that of 24hLPS/DCs (fully mature), and greater than that of 0hLPS/DCs (immature). A functional assay showed that 4hLPS/DCs displayed increased endocytotic ability compared to 24hLPS/DCs, indicating a semi-mature state. 4hLPS/DCs were greater producers of IL-10 protein and TGFbeta1 mRNA than 24hLPS/DCs and immature DCs, displaying a cytokine production pattern that is characteristic of tolerogenic DCs. An assay for antigen-presenting capacity demonstrated that 4hLPS/DCs induced secretion of IL-2 from an OTH4 T cell hybridoma, indicating a functional presenting activity. Finally, the tolerogenic phenotype of 4hLPS/DCs was demonstrated by their ability to interfere with the progression of bovine type II collagen (bII)-induced arthritis (CIA) when they were loaded with bCII antigen and injected into mice with established CIA. We conclude that the stimulation of murine bone marrow-derived DCs with LPS for four hours generates semi-mature DCs with tolerogenic capability.

Multiple imputation procedures allow the rescue of missing data: an application to determine serum tumor necrosis factor (TNF) concentration values during the treatment of rheumatoid arthritis patients with anti-TNF therapy.

Longitudinal studies aimed at evaluating patients clinical response to specific therapeutic treatments are frequently summarized in incomplete datasets due to missing data. Multivariate statistical procedures use only complete cases, deleting any case with missing data. MI and MIANALYZE procedures of the SAS software perform multiple imputations based on the Markov Chain Monte Carlo method to replace each missing value with a plausible value and to evaluate the efficiency of such missing data treatment. The objective of this work was to compare the evaluation of differences in the increase of serum TNF concentrations depending on the -308 TNF promoter genotype of rheumatoid arthritis (RA) patients receiving anti-TNF therapy with and without multiple imputations of missing data based on mixed models for repeated measures. Our results indicate that the relative efficiency of our multiple imputation model is greater than 98% and that the related inference was significant (p-value < 0.001). We established that under both approaches serum TNF levels in RA patients bearing the G/A -308 TNF promoter genotype displayed a significantly (p-value < 0.0001) increased ability to produce TNF over time than the G/G patient group, as they received successively doses of anti-TNF therapy.

Multiple imputation procedures allow the rescue of missing data: An application to determine serum tumor necrosis factor (TNF) concentration values during the treatment of rheumatoid arthritis patients with anti-TNF therapy

Longitudinal studies aimed at evaluating patients clinical response to specific therapeutic treatments are frequently summarized in incomplete datasets due to missing data. Multivariate statistical procedures use only complete cases, deleting any case with missing data. MI and MIANALYZE procedures of the SAS software perform multiple imputations based on the Markov Chain Monte Carlo method to replace each missing value with a plausible value and to evaluate the efficiency of such missing data treatment. The objective of this work was to compare the evaluation of differences in the increase of serum TNF concentrations depending on the ¡308 TNF promoter genotype of rheumatoid arthritis (RA) patients receiving anti-TNF therapy with and without multiple imputations of missing data based on mixed models for repeated measures. Our results indicate that the relative efficiency of our multiple imputation model is greater than 98 percent and that the related inference was significant (p-value < 0.001). We established that under both approaches serum TNF levels in RA patients bearing the G/A ¡308 TNF promoter genotype displayed a significantly (p-value < 0.0001) increased ability to produce TNF over time than the G/G patient group, as they received successively doses of anti-TNF therapy.

A rare haplotype of the RET proto-oncogene is a risk-modifying allele in hirschsprung disease.

Hirschsprung disease (HSCR) is a common genetic disorder characterized by intestinal obstruction secondary to enteric aganglionosis. HSCR demonstrates a complex pattern of inheritance, with the RET proto-oncogene acting as a major gene and with several additional susceptibility loci related to the Ret-signaling pathway or to other developmental programs of neural crest cells. To test how the HSCR phenotype may be affected by the presence of genetic variants, we investigated the role of a single-nucleotide polymorphism (SNP), 2508C-->T (S836S), in exon 14 of the RET gene, characterized by low frequency among patients with HSCR and overrepresentation in individuals affected by sporadic medullary thyroid carcinoma. Typing of several different markers across the RET gene demonstrated that a whole conserved haplotype displayed anomalous distribution and nonrandom segregation in families with HSCR. We provide genetic evidence about a protective role of this low-penetrant haplotype in the pathogenesis of HSCR and demonstrate a possible functional effect linked to RET messenger RNA expression.