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.     

The Interplay Between NSAIDs and Candida albicans on the Gastrointestinal Tract of Guinea Pigs

Recent studies suggest that Candida albicans colonization is associated with several gastrointestinal inflammatory disorders and is also responsible for the delay in ulcer healing. No data are reported about the effects of C. albicans on the nonsteroidal anti-inflammatory drugs (NSAIDs)-induced necroinflammatory lesions. On the other hand, beneficial effects of NSAIDs regarding the colonization potential with C. albicans have been reported. Our aim was to investigate whether the association between NSAIDs and C. albicans could potentially induce necroinflammatory lesions in the guinea pigs gastric and enteral mucosa. Three interventional groups of 11 guinea pigs each were investigated after 5 days of receiving indomethacin, C. albicans or the association of both. C. albicans and necroinflammatory lesions were graded based on histological examinations. Statistical analysis used Mann–Whitney nonparametric test. NSAIDs did not significantly decrease C. albicans colonization grades on gastrointestinal mucosa. Administration of indomethacin subsequent to C. albicans determined significantly more severe necroinflammatory lesions compared to group that only received C. albicans. The association of NSAIDs and C. albicans did not cause significantly more severe degenerative or inflammatory lesions compared to the administration of only NSAIDs in this experimental model. Associations between NSAIDs and C. albicans caused significantly more severe necroinflammatory injuries than the lesions produced by C. albicans, without enhancing the mucosal injury or inflammation caused by NSAIDs.     

Deep brain stimulation

During the last decade deep brain stimulation (DBS) has become a routine method for the treatment of advanced Parkinsons disease (PD), leading to striking improvements in motor function and quality of life of PD patients. It is associated with minimal morbidity. The rationale of targeting specific structures within basal ganglia such as the subthalamic nucleus (STN) or the internal segment of the globus pallidus (GPi) is strongly supported by the current knowledge of the basal ganglia pathophysiology, which is derived from extensive experimental work and which provides the theoretical basis for surgical therapy in PD. In particular, the STN has advanced to the worldwide most used target for DBS in the treatment of PD, due to the marked improvement of all cardinal symptoms of the disease. Moreover on-period dyskinesias are reduced in parallel with a marked reduction of the equivalent daily levodopa dose following STN–DBS. The success of the therapy largely depends on the selection of the appropriate candidate patients and on the precise implantation of the stimulation electrode, which necessitates careful imaging-based pre-targeting and extensive electrophysiological exploration of the target area. Despite the clinical success of the therapy, the fundamental mechanisms of high-frequency stimulation are still not fully elucidated. There is a large amount of evidence from experimental and clinical data that stimulation frequency represents a key factor with respect to clinical effect of DBS. Interestingly, high-frequency stimulation mimics the functional effects of ablation in various brain structures. The main hypotheses for the mechanism of high-frequency stimulation are: (1) depolarization blocking of neuronal transmission through inactivation of voltage dependent ion-channels, (2) jamming of information by imposing an efferent stimulation-driven high-frequency pattern, (3) synaptic inhibition by stimulation of inhibitory afferents to the target nucleus, (4) synaptic failure by stimulation-induced neurotransmitter depletion. As the hyperactivity of the STN is considered a functional hallmark of PD and as there is experimental evidence for STN-mediated glutamatergic excitotoxicity on neurons of the substantia nigra pars compacta (SNc), STN–DBS might reduce glutamatergic drive, leading to neuroprotection. Further studies will be needed to elucidate if STN–DBS indeed provides a slow-down of disease progression.     

Accuracy of cDNA microarray methods to detect small gene expression changes induced by neuregulin on breast epithelial cells

Background  

cDNA microarrays are a powerful means to screen for biologically relevant gene expression changes, but are often limited by their ability to detect small changes accurately due to "noise" from random and systematic errors. While experimental designs and statistical analysis methods have been proposed to reduce these errors, few studies have tested their accuracy and ability to identify small, but biologically important, changes. Here, we have compared two cDNA microarray experimental design methods with northern blot confirmation to reveal changes in gene expression that could contribute to the early antiproliferative effects of neuregulin on MCF10AT human breast epithelial cells.     

Gene expression databases and data mining

The DNA microarray technology has arguably caught the attention of the worldwide life science community and is now systematically supporting major discoveries in many fields of study. The majority of the initial technical challenges of conducting experiments are being resolved, only to be replaced with new informatics hurdles, including statistical analysis, data visualization, interpretation, and storage. Two systems of databases, one containing expression data and one containing annotation data are quickly becoming essential knowledge repositories of the research community. This present paper surveys several databases, which are considered "pillars" of research and important nodes in the network. This paper focuses on a generalized workflow scheme typical for microarray experiments using two examples related to cancer research. The workflow is used to reference appropriate databases and tools for each step in the process of array experimentation. Additionally, benefits and drawbacks of current array databases are addressed, and suggestions are made for their improvement.     

Proliferation and competition in discrete biological systems

We study the emergence of collective spatio-temporal objects in biological systems by representing individually the elementary interactions between their microscopic components. We use the immune system as a prototype for such interactions. The results of this detailed explicit analysis are compared with the traditional procedure of representing the collective dynamics in terms of densities that obey partial differential equations. The simulations show even for very simple elementary reactions the spontaneous emergence of localized complex structures, from microscopic noise. In turn the effective dynamics of these structures affects the average behaviour of the system in a very decisive way: systems which would according to the differential equations approximation die, display in reality a very lively behaviour. As the optimal modelling method we propose a mixture of microscopic simulation systems describing each reaction separately, and continuous methods describing the average behaviour of the agents.     

Endocardial Tip Cells in the Human Embryo – Facts and Hypotheses

Experimental studies regarding coronary embryogenesis suggest that the endocardium is a source of endothelial cells for the myocardial networks. As this was not previously documented in human embryos, we aimed to study whether or not endothelial tip cells could be correlated with endocardial-dependent mechanisms of sprouting angiogenesis. Six human embryos (43–56 days) were obtained and processed in accordance with ethical regulations; immunohistochemistry was performed for CD105 (endoglin), CD31, CD34, α-smooth muscle actin, desmin and vimentin antibodies. Primitive main vessels were found deriving from both the sinus venosus and aorta, and were sought to be the primordia of the venous and arterial ends of cardiac microcirculation. Subepicardial vessels were found branching into the outer ventricular myocardium, with a pattern of recruiting α-SMA+/desmin+ vascular smooth muscle cells and pericytes. Endothelial sprouts were guided by CD31+/CD34+/CD105+/vimentin+ endothelial tip cells. Within the inner myocardium, we found endothelial networks rooted from endocardium, guided by filopodia-projecting CD31+/CD34+/CD105+/ vimentin+ endocardial tip cells. The myocardial microcirculatory bed in the atria was mostly originated from endocardium, as well. Nevertheless, endocardial tip cells were also found in cardiac cushions, but they were not related to cushion endothelial networks. A general anatomical pattern of cardiac microvascular embryogenesis was thus hypothesized; the arterial and venous ends being linked, respectively, to the aorta and sinus venosus. Further elongation of the vessels may be related to the epicardium and subepicardial stroma and the intramyocardial network, depending on either endothelial and endocardial filopodia-guided tip cells in ventricles, or mostly on endocardium, in atria.     

Cytokine profiles of HeLa and human diploid cells induced by different fractions of Vibrio parahaemolyticus cultures exposed to stress conditions

Vibrio (V.) parahaemolyticus is an aquatic halophilic bacteria which produces gastroenteritis and in rare cases septicaemia after the consumption of raw or under-cooked contaminated seafood.The severity of diarrheal illness caused by this bacterium is closely related to the presence of two types of hemolysins (the thermostable direct hemolysin-TDH and TDH related hemolysin-TRH) and also of type III secretion system (TTSS) proteins. The TTSS type 1 induces a wide array of effects on infected HeLa cells such as autophagy, oncosis, cell rounding and lysis. Previous studies have shown that heat shock proteins have the ability to stimulate the production of interleukins in different cellular cultures. In our studies we have stimulated two cellular lines (HeLa and human diploid cells) with different V. parahaemolyticus culture fractions in order to observe the effect on cytokines production. Thus, the purpose of this study was to analyze the expression of IL-1, IL-2, IL-4, IL-6, IL-10 and TNF-alpha induced by the cell treatment with total cellular lysate, periplasmic fractions and culture supernatants extracted from V. parahaemolyticus exposed to normal and also to stress conditions. The ELISA assay of the cytokine profile of the HeLa and HDC cell lines stimulated with different bacterial fractions revealed that in the V. parahemolyticus cultures submitted to osmotic and heat shock stress are accumulating factors (probably heat shock proteins) which are exhibiting immunomodulatory activity, responsible for the induction of a pro-inflammatory response associated with increased levels of IL-6 and TNF-alpha expression, however balanced by the stimulation of the anti-inflammatory cytokine IL-4 synthesis.