Applications of Genome-Editing Technologies for Type 1 Diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the destruction of insulin-producing pancreatic ß-cells by the immune system. Although conventional therapeutic modalities, such as insulin injection, remain a mainstay, recent years have witnessed the emergence of novel treatment approaches encompassing immunomodulatory therapies, such as stem cell and ß-cell transplantation, along with revolutionary gene-editing techniques. Notably, recent research endeavors have enabled the reshaping of the T-cell repertoire, leading to the prevention of T1D development. Furthermore, CRISPR-Cas9 technology has demonstrated remarkable potential in targeting endogenous gene activation, ushering in a promising avenue for the precise guidance of mesenchymal stem cells (MSCs) toward differentiation into insulin-producing cells. This innovative approach holds substantial promise for the treatment of T1D. In this review, we focus on studies that have developed T1D models and treatments using gene-editing systems.

Dendritic cell activation and screening for key molecular signatures required for the induction of allergic responses.

The chain of events that leads to the sensitization of the immune system to environmental antigens, resulting in the onset of allergic disease, has been studied in great detail over the past 30 years. However, during this time, the rate of allergic diseases has increased exponentially, indicating the need to concentrate our studies on host-environmental factors that contribute to the onset of disease. Monocyte-derived dendritic cells (DCs) play a key role in driving localized and systemic immune responses. In this study, we developed a platform for screening the molecular signature and phenotypic profile of DCs activated by allergenic stimuli, including TSLP, IL-25, IL-33, IL-1a, Vit-D3 (1α,25-Dihydroxyvitamin D3), PAR1-AP Peptide, Papain, and recombinant human DerP1 protein to induce a type II associated inflammatory signature. Following activation with allergenic stimuli, modulated DCs are subjected to deep phenotyping via flow cytometry for surface and intracellular markers to detect and/or validate immunomodulatory properties. RNA sequencing is further used to compare the gene expression profiles of DCs responding to either allergenic or microbial stimuli, including the TLR3 agonist dsRNA Poly I:C and TLR4 agonist LPS. In our study, we aimed to identify key molecular signatures of DCs involved in the development of asthma and allergy based on their comparative activation with this broad panel of allergens. We expect to determine central control modules of transcription factors in DCs associated with Th2 induction.

Gut microbial influences on the adaptive immune system and the development of cow milk allergy.

Allergic diseases constitute significant health and economic issues in both developed and developing nations, with epidemiological studies demonstrating a rapid increase in the global prevalence of food allergy among the pediatric population. Cow milk protein allergy (CMPA), one of the most common forms of food allergies observed in early childhood, affects between 2%-6% of infants and children under 3 years of age. CMPA can present as either an IgE-mediated atopic allergy or a non-IgE mediated allergic response. Antigen-specific T cells play a pivotal role in directing the type of inflammatory immune response that occurs as well as in the formation of immunological memory. IgE-mediated CMPA is thought to develop because of an abnormal expansion of allergen-specific type-2 helper T (Th2) cells and a corresponding deficiency in immune regulation by regulatory T cells (Tregs), thereby altering the Th2/Treg balance. The gut microbiota, established very early during childhood through host-microbe interactions, can influence the incidence of allergic diseases. In this study, we aimed to analyze both the microbiome composition and CD4+T cell differentiation patterns in pediatric patients with and without cow milk allergy to establish the association between these factors. Using 16S rRNA sequencing, we analyzed the microbiome composition in stool samples of allergic and non-allergic pediatric patients aged between 1-4 years and identified the microbial species abundant in IgE and non-IgE mediated cow milk allergies. To assess the CD4+T cell differentiation patterns, peripheral blood mononuclear cells (PBMCs) from these patients were re-stimulated with cow milk antigen, and T cell subsets were assessed using flow cytometry. Antigen-specific CD4+T cells were identified and sorted for high throughput sequencing and subsequent gene expression analysis. The CD4+T cell differentiation patterns of the total and antigen-specific T cells were analyzed and statistically compared with controls. The identification of the correlation between the CD4+T cell differentiation patterns and species-specific microbial abundance in IgE and non-IgE mediated cow milk allergies can help in determining how the gut microbiome influences the CD4+T cell immune compartment development, ultimately leading to the development of cow milk allergy in pediatric patients.

Anti-proliferative and anti-inflammatory activities of the sea cucumber Holothuria polii aqueous extract.

OBJECTIVE: Sea cucumbers are considered among the most important functional foods. Following bioassay guided fractionation, we assessed the anti-proliferative and anti-inflammatory activities of Holothuria polii (H. polii) extracts. METHODS: Sea cucumber ethanolic extract and the partially purified aqueous fractions were assessed for their anti-proliferative activities. These latter bioactivities were evaluated in the highly invasive MDA-MB-231 human breast cancer cells in two-dimensional and three-dimensional cultures using trypan blue exclusion assay. The tumor-suppressive effects of sea cucumber ethanolic extract and aqueous fractions were assayed by measuring the trans-well invasion of MDA-MB-231 cells and the expression of some epithelial mesenchymal transition markers using quantitative reverse-transcription polymerase chain reaction and western blot analysis. The anti-inflammatory activity of the aqueous fraction was tested by measuring the secreted levels of interleukin-6, nitric oxide, and matrix metalloproteinase 9 in endotoxin-induced mammary epithelial SCp2 cells and interleukin-1ß in phorbol-12-myristate-13-acetate-activated human monocytic THP-1 cells. RESULTS: Sea cucumber ethanolic extract and the aqueous fraction significantly decreased the proliferation of MDA-MB-231 cells by more than 50% at similar and noncytotoxic concentrations and caused an arrest in the S-phase of the cell cycle of treated cells. In contrast, petroleum ether, chloroform, ethyl acetate, and n-butanol organic fractions did not show any significant activity. Furthermore, sea cucumber ethanolic extract and aqueous fraction reduced the proliferation of MDA-MB-231 cells in three-dimensional cultures by more than 60% at noncytotoxic concentrations. In addition, treatment with these concentrations resulted in the loss of stellate outgrowths in favor of spherical aggregates and a 30% decrease in invasive properties. Both sea cucumber ethanolic extract and aqueous decreased the transcription of vimentin and the protein expression levels of vimentin and N-cadherin in three-dimensional cultures. The aqueous fraction decreased the levels of inflammatory markers interleukin-6, nitric oxide, and matrix metalloproteinase 9 in the mouse mammary SCp2 cells, and the level of interleukin-1ß produced by phorbol-12-myristate-13-acetate-activated THP-1 human monocytic cells. CONCLUSION: The data reveal for the first time promising anti-proliferative and anti-inflammatory activities in H. polii water extract in two-dimensional and three-dimensional culture models.