Design and preclinical testing of an anti-CD41 CAR T cell for the treatment of acute megakaryoblastic leukaemia

Acute megakaryoblastic leukaemia (AMkL) is a rare subtype of acute myeloid leukaemia (AML) representing 5% of all reported cases, and frequently diagnosed in children with Down syndrome. Patients diagnosed with AMkL have low overall survival and have poor outcome to treatment, thus novel therapies such as CAR T cell therapy could represent an alternative in treating AMkL. We investigated the effect of a new CAR T cell which targets CD41, a specific surface antigen for M7-AMkL, against an in vitro model for AMkL, DAMI Luc2 cell line. The performed flow cytometry evaluation highlighted a percentage of 93.8% CAR T cells eGFP-positive and a limited acute effect on lowering the target cell population. However, the interaction between effector and target (E:T) cells, at a low ratio, lowered the cell membrane integrity, and reduced the M7-AMkL cell population after 24 h of co-culture, while the cytotoxic effect was not significant in groups with higher E:T ratio. Our findings suggest that the anti-CD41 CAR T cells are efficient for a limited time spawn and the cytotoxic effect is visible in all experimental groups with low E:T ratio.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

Mesenchymal-to-Endothelial Transition in Kaposi Sarcoma: A Histogenetic Hypothesis Based on a Case Series and Literature Review

Objectives

Although several studies have been conducted regarding Kaposi sarcoma (KS), its histogenesis still remains to be elucidated. The aim of our study was to analyze the immunophenotype of Kaposi sarcoma and to present a hypothesis about the histogenesis of this tumor, based on a case series and a review of relevant literature.

Methods

In 15 cases of KSs diagnosed during 2000–2011, the clinicopathological features were correlated with the immunoexpression of c-Kit, SMA, CD34, CD31, vascular endothelial growth factor (VEGF), COX-2, c-KIT, smooth muscle antigen (SMA), and stem cell surface marker CD105.

Results

Both CD105 and c-KIT rate of the spindle-shaped tumor cell positivity increased in parallel to the pathological stage. All cases displayed CD105 and weak c-KIT positivity in the endothelial cells. SMA, VEGF, and COX-2 were focally expressed in all cases. CD34 marked both endothelium and spindle-shaped tumor cells. No c-KIT expression was noticed in KS of the internal organs.

Conclusions

KS seems to be a variant of myofibroblastic tumors that originates from the viral modified pluripotent mesenchymal cells of the connective tissue transformed in spindle-shaped KS cells, followed by a mesenchymal-endothelial transition and a myofibroblastic-like differentiation. This paper mailnly showed that KS cannot be considered a pure vascular tumor.     

Anti‐atherosclerotic effects of vitamin E – myth or reality?

Atherosclerosis and its complications such as coronary heart disease, myocardial infarction and stroke are the leading causes of death in the developed world. High blood pressure, diabetes, smoking and a diet high in cholesterol and lipids clearly increase the likelihood of premature atherosclerosis, albeit other factors, such as the individual genetic makeup, may play an additional role. Several epidemiological studies and intervention trials have been performed with vitamin E, and some of them showed that it prevents atherosclerosis. For a long time, vitamin E was assumed to act by decreasing the oxidation of LDL, a key step in atherosclerosis initiation. However, at the cellular level, vitamin E acts by inhibition of smooth muscle cell proliferation, platelet aggregation, monocyte adhesion, oxLDL uptake and cytokine production, all reactions implied in the progression of atherosclerosis. Recent research revealed that these effects are not the result of the antioxidant activity of vitamin E, but rather of precise molecular actions of this compound. It is assumed that specific interactions of vitamin E with enzymes and proteins are at the basis of its non-antioxidant effects. Vitamin E influences the activity of several enzymes (e.g. PKC, PP2A, COX-2, 5-lipooxygenase, nitric oxide synthase, NADPH-oxidase, superoxide dismutase, phopholipase A2) and modulates the expression of genes that are involved in atherosclerosis (e.g. scavenger receptors, integrins, selectins, cytokines, cyclins). These interactions promise to reveal the biological properties of vitamin E and allow designing better strategies for the protection against atherosclerosis progression.     

Dietary input of microbes and host genetic variation shape among-population differences in stickleback gut microbiota

To explain differences in gut microbial communities we must determine how processes regulating microbial community assembly (colonization, persistence) differ among hosts and affect microbiota composition. We surveyed the gut microbiota of threespine stickleback (Gasterosteus aculeatus) from 10 geographically clustered populations and sequenced environmental samples to track potential colonizing microbes and quantify the effects of host environment and genotype. Gut microbiota composition and diversity varied among populations. These among-population differences were associated with multiple covarying ecological variables: habitat type (lake, stream, estuary), lake geomorphology and food- (but not water-) associated microbiota. Fish genotype also covaried with gut microbiota composition; more genetically divergent populations exhibited more divergent gut microbiota. Our results suggest that population level differences in stickleback gut microbiota may depend more on internal sorting processes (host genotype) than on colonization processes (transient environmental effects).     

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

MIND-BEST: Web server for drugs and target discovery; design, synthesis, and assay of MAO-B inhibitors and theoretical-experimental study of G3PDH protein from Trichomonas gallinae

Many drugs with very different affinity to a large number of receptors are described. Thus, in this work, we selected drug-target pairs (DTPs/nDTPs) of drugs with high affinity/nonaffinity for different targets. Quantitative structure-activity relationship (QSAR) models become a very useful tool in this context because they substantially reduce time and resource-consuming experiments. Unfortunately, most QSAR models predict activity against only one protein target and/or they have not been implemented on a public Web server yet, freely available online to the scientific community. To solve this problem, we developed a multitarget QSAR (mt-QSAR) classifier combining the MARCH-INSIDE software for the calculation of the structural parameters of drug and target with the linear discriminant analysis (LDA) method in order to seek the best model. The accuracy of the best LDA model was 94.4% (3,859/4,086 cases) for training and 94.9% (1,909/2,012 cases) for the external validation series. In addition, we implemented the model into the Web portal Bio-AIMS as an online server entitled MARCH-INSIDE Nested Drug-Bank Exploration & Screening Tool (MIND-BEST), located at http://miaja.tic.udc.es/Bio-AIMS/MIND-BEST.php . This online tool is based on PHP/HTML/Python and MARCH-INSIDE routines. Finally, we illustrated two practical uses of this server with two different experiments. In experiment 1, we report for the first time a MIND-BEST prediction, synthesis, characterization, and MAO-A and MAO-B pharmacological assay of eight rasagiline derivatives, promising for anti-Parkinson drug design. In experiment 2, we report sampling, parasite culture, sample preparation, 2-DE, MALDI-TOF and -TOF/TOF MS, MASCOT search, 3D structure modeling with LOMETS, and MIND-BEST prediction for different peptides as new protein of the found in the proteome of the bird parasite Trichomonas gallinae, which is promising for antiparasite drug targets discovery.     

The immunomodulatory protein B7-H4 is overexpressed in breast and ovarian cancers and promotes epithelial cell transformation

B7-H4 protein is expressed on the surface of a variety of immune cells and functions as a negative regulator of T cell responses. We independently identified B7-H4 (DD-O110) through a genomic effort to discover genes upregulated in tumors and here we describe a new functional role for B7-H4 protein in cancer. We show that B7-H4 mRNA and protein are overexpressed in human serous ovarian cancers and breast cancers with relatively little or no expression in normal tissues. B7-H4 protein is extensively glycosylated and displayed on the surface of tumor cells and we provide the first demonstration of a direct role for B7-H4 in promoting malignant transformation of epithelial cells. Overexpression of B7-H4 in a human ovarian cancer cell line with little endogenous B7-H4 expression increased tumor formation in SCID mice. Whereas overexpression of B7-H4 protected epithelial cells from anoikis, siRNA-mediated knockdown of B7-H4 mRNA and protein expression in a breast cancer cell line increased caspase activity and apoptosis. The restricted normal tissue distribution of B7-H4, its overexpression in a majority of breast and ovarian cancers and functional activity in transformation validate this cell surface protein as a new target for therapeutic intervention. A therapeutic antibody strategy aimed at B7-H4 could offer an exciting opportunity to inhibit the growth and progression of human ovarian and breast cancers.