ID2-ETS2 axis regulates the transcriptional acquisition of pro-tumoral microglia phenotype in glioma.

Glioblastoma is a highly aggressive brain tumour that creates an immunosuppressive microenvironment. Microglia, the brain's resident immune cells, play a crucial role in this environment. Glioblastoma cells can reprogramme microglia to create a supportive niche that promotes tumour growth. However, the mechanisms controlling the acquisition of a transcriptome associated with a tumour-supportive microglial reactive state are not fully understood. In this study, we investigated changes in the transcriptional profile of BV2 microglia exposed to C6 glioma cells. RNA-sequencing analysis revealed a significant upregulation of microglial inhibitor of DNA binding 1 (Id1) and Id2, helix-loop-helix negative transcription regulatory factors. The concomitant regulation of microglial ETS proto-oncogene 2, transcription factor (ETS2)-target genes, i.e., Dusp6, Fli1, Jun, Hmox1, and Stab1, led us to hypothesize that ETS2 could be regulated by ID proteins. In fact, ID2-ETS2 protein interactions increased in microglia exposed to glioma cells. In addition, perturbation of the ID2-ETS2 transcriptional axis influenced the acquisition of a microglial tumour-supportive phenotype. ID2 and ETS2 genes were found to be expressed by the tumour-associated microglia isolated from human glioblastoma tumour biopsies. Furthermore, ID2 and ETS2 gene expressions exhibited inverse prognostic values in patients with glioma in cohorts from The Cancer Genome Atlas. Collectively, our findings indicate that the regulation of ETS2 by ID2 plays a role in the transcriptional regulation of microglia in response to stimuli originating from glioblastoma cells, information that could lead to developing therapeutic strategies to manipulate microglial tumour-trophic functions.

Effects of buffer composition and plasmid toxicity on electroporation-based non-viral gene delivery in mammalian cells using bursts of nanosecond and microsecond pulses.

Gene electrotransfer (GET) is non-viral gene delivery technique, also known as electroporation-mediated gene delivery or electrotransfection. GET is a method used to introduce foreign genetic material (such as DNA or RNA) into cells by applying external pulsed electric fields (PEFs) to create temporary pores in the cell membrane. This study was undertaken to examine the impact of buffer composition on the efficiency of GET in mammalian cells Also, we specifically compared the effectiveness of high-frequency nanosecond (ns) pulses with standard microsecond (µs) pulses. For the assessment of cell transfection efficiency and viability, flow cytometric analysis, luminescent assays, and measurements of metabolic activity were conducted. The efficiency of electrotransfection was evaluated using two different proteins encoding plasmids (pEGFP-N1 and Luciferase-pcDNA3). The investigation revealed that the composition of the electroporation buffer significantly influences the efficacy of GET in CHO-K1 cell line. The different susceptibility of cell lines to the electric field and the plasmid cytotoxicity were reported. It was also shown that electroporation with nanosecond duration PEF protocols ensured equivalent or even better transfection efficiency than standard µsPEF. Additionally, we successfully performed long-term transfection of the murine 4T1 cell line using high-frequency nanosecond PEFs and confirmed its' applicability in an in vivo model. The findings from the study can be applied to optimize electrotransfection conditions.

Laboratory mice with a wild microbiota generate strong allergic immune responses.

Allergic disorders are caused by a combination of hereditary and environmental factors. The hygiene hypothesis postulates that early-life microbial exposures impede the development of subsequent allergic disease. Recently developed "wildling" mice are genetically identical to standard laboratory specific pathogen-free (SPF) mice but are housed under seminatural conditions and have rich microbial exposures from birth. Thus, by comparing conventional SPF mice with wildlings, we can uncouple the impact of lifelong microbial exposures from genetic factors on the allergic immune response. We found that wildlings developed larger populations of antigen-experienced T cells than conventional SPF mice, which included interleukin-10-producing CD4 T cells specific for commensal Lactobacilli strains and allergy-promoting T helper 2 (TH2) cells. In models of airway exposure to house dust mite (HDM), recombinant interleukin-33, or Alternaria alternata, wildlings developed strong allergic inflammation, characterized by eosinophil recruitment, goblet cell metaplasia, and antigen-specific immunoglobulin G1 (IgG1) and IgE responses. Wildlings developed robust de novo TH2 cell responses to incoming allergens, whereas preexisting TH2 cells could also be recruited into the allergic immune response in a cytokine-driven and TCR-independent fashion. Thus, wildling mice, which experience diverse and lifelong microbial exposures, were not protected from developing pathological allergic immune responses. Instead, wildlings mounted robust allergic responses to incoming allergens, shedding new light on the hygiene hypothesis.

Understanding the Immunomodulatory Effects of Bovine Colostrum: Insights into IL-6/IL-10 Axis-Mediated Inflammatory Control.

Bovine colostrum (COL), the first milk secreted by lactating cows postpartum, is a rich source of bioactive compounds that exert a significant role in the survival, growth, and immune development of neonatal calves. This study investigated the immunomodulatory effects of COL on cytokine production in vitro using a Caco-2/THP-1 macrophage co-culture model stimulated with Phorbol 12-myristate 13-acetate (PMA). COL pretreatment significantly reduced IL-6 (241.3 pg/mL) production induced by PMA (p < 0.05), while increasing IL-10 production (45.3 pg/mL), in comparison to PMA control (441.1 and 12.5 pg/mL, respectively). Further investigations revealed that the IL-6 suppressive effect of colostrum was heat-sensitive and associated with components of higher molecular mass (100 kDa). Moreover, colostrum primarily influenced THP-1 macrophages rather than Caco-2 epithelial cells. The effects of colostrum on IL-6 production were associated with reduced NF-κB activation in THP-1 macrophages. In calf-FMT transplanted C57BL/6 murine model, colostrum decreased intestinal permeability, reduced immune cell infiltration and intestinal score, and suppressed IL-6 (142.0 pg/mL) production during S. typhimurium infection, in comparison to control animals (215.2 pg/mL). These results suggest the immunomodulatory activity of bovine colostrum and its potential applications in inflammatory disorders. Further studies are needed to elucidate the underlying mechanisms and validate the findings in bovine models.

The Influence of Feeding with Colostrum and Colostrum Replacer on Major Blood Biomarkers and Growth Performance in Dairy Calves.

Bovine colostrum (BC) is the first milk produced by lactating cows after parturition. BC is rich in various amino acids, proteins, and fats essential for the nutrition of the neonate calves. Despite the evident beneficial effect of BC on calves, the effect of BC on blood biomarkers is poorly understood. Calves that received BC showed significantly higher body mass at days 7 and 30 (38.54 kg and 43.42 kg, respectively) compared to the colostrum replacer group (p = 0.0064). BC induced greater quantities of blood neutrophils (0.27 × 109/L) and monocytes (4.76 × 109/L) in comparison to the colostrum replacer (0.08 and 0.06 × 109/L, respectively) (p = 0.0001). Animals that received BC showed higher levels of total serum protein (59.16 g/L) and albumin (29.96 g/L) in comparison to the colostrum replacer group (44.34 g/L and 31.58 g/L, respectively). In addition, BC induced greater intestinal mucus production in the Wistar rat model. Collectively, these results demonstrate that BC is important for the growth of calves and that it provides a significant beneficial effect on morphological and biochemical blood parameters.

High-Frequency Nanosecond Bleomycin Electrochemotherapy and its Effects on Changes in the Immune System and Survival.

In this work, a time-dependent and time-independent study on bleomycin-based high-frequency nsECT (3.5 kV/cm × 200 pulses) for the elimination of LLC1 tumours in C57BL/6J mice is performed. We show the efficiency of nsECT (200 ns and 700 ns delivered at 1 kHz and 1 MHz) for the elimination of tumours in mice and increase of their survival. The dynamics of the immunomodulatory effects were observed after electrochemotherapy by investigating immune cell populations and antitumour antibodies at different timepoints after the treatment. ECT treatment resulted in an increased percentage of CD4+ T, splenic memory B and tumour-associated dendritic cell subsets. Moreover, increased levels of antitumour IgG antibodies after ECT treatment were detected. Based on the time-dependent study results, nsECT treatment upregulated PD 1 expression on splenic CD4+ Tr1 cells, increased the expansion of splenic CD8+ T, CD4+CD8+ T, plasma cells and the proportion of tumour-associated pro inflammatory macrophages. The Lin- population of immune cells that was increased in the spleens and tumour after nsECT was identified. It was shown that nsECT prolonged survival of the treated mice and induced significant changes in the immune system, which shows a promising alliance of nanosecond electrochemotherapy and immunotherapy.

Effects of Time Delay Between Unipolar Pulses in High Frequency Nano-Electrochemotherapy.

OBJECTIVE: this work focuses on bleomycin electrochemotherapy using new modality of high repetition frequency unipolar nanosecond pulses. METHODS: As a tumor model, Lewis lung carcinoma (LLC1) cell line in C57BL mice (n = 42) was used. Electrochemotherapy was performed with intertumoral injection of bleomycin (50 µL of 1500 IU solution) followed by nanosecond and microsecond range electrical pulse delivery via parallel plate electrodes. The 3.5 kV/cm pulses of 200 and 700 ns were delivered in a burst of 200 at frequencies of 1 kHz and 1 MHz. For comparison of treatment efficiency, a standard 1.3 kV/cm x 100 µs x 8 protocol was used. RESULTS: It was shown that it is possible to manipulate the efficacy of unipolar sub-microsecond electrochemotherapy solely by the time delay between the pulses. SIGNIFICANCE: the results suggest that the sub-microsecond range pulses can be as effective as the protocols in European Standard Operating Procedures on Electrochemotherapy (ESOPE) using 100 µs pulses.

Electrochemotherapy Using Doxorubicin and Nanosecond Electric Field Pulses: A Pilot in Vivo Study.

Pulsed electric field (PEF) is frequently used for intertumoral drug delivery resulting in a well-known anticancer treatment-electrochemotherapy. However, electrochemotherapy is associated with microsecond range of electrical pulses, while nanosecond range electrochemotherapy is almost non-existent. In this work, we analyzed the feasibility of nanosecond range pulse bursts for successful doxorubicin-based electrochemotherapy in vivo. The conventional microsecond (1.4 kV/cm × 100 µs × 8) procedure was compared to the nanosecond (3.5 kV/cm × 800 ns × 250) non-thermal PEF-based treatment. As a model, Sp2/0 tumors were developed. Additionally, basic current and voltage measurements were performed to detect the characteristic conductivity-dependent patterns and to serve as an indicator of successful tumor permeabilization both in the nano and microsecond pulse range. It was shown that nano-electrochemotherapy can be the logical evolution of the currently established European Standard Operating Procedures for Electrochemotherapy (ESOPE) protocols, offering better energy control and equivalent treatment efficacy.

Sub-microsecond electrotransfection using new modality of high frequency electroporation.

Micro-millisecond range electric field pulses have been used for decades to facilitate DNA transfer into cells and tissues, while the growing number of clinical trials underline the strong potential of DNA electroporation. In this work, we present new sub-microsecond range protocols and methodology enabling successful electrotransfection in the sub-microsecond range. To facilitate DNA transfer, a 3 kV/60 A and high frequency (1 MHz) sub-microsecond range square wave generator was applied in the study. As a model, Chinese hamster ovary (CHO-K1) cells were used. Sub-microsecond range (300-700 ns) high frequency pulsed electric fields of 2-15 kV/cm were applied. The efficiency of electrotransfection was evaluated using two green fluorescent protein encoding plasmids of different size (3.5 kbp and 4.7 kbp). It was shown that transfection efficiency cannot be effectively improved with increase of the number of pulses after a certain threshold, however, independently on the plasmid size, the proposed sub-microsecond range pulsing methodology (2-5 kV/cm; n = 250) efficiency-wise was equivalent to 1.5 kV/cm × 100 µs × 4 electroporation procedure. The results of the study are useful for further development of in vitro and in vivo methods for effective electrotransfer of DNA using shorter pulses.