Sublingual Immunization with Chimeric C1q/CD40 Ligand/HIV Virus-like Particles Induces Strong Mucosal Immune Responses against HIV

Development of a vaccine that can elicit robust HIV specific antibody responses in the mucosal compartments is desired for effective prevention of HIV via sexual transmission. However, the current mucosal vaccines have either poor immunogenicity when administered orally or invite safety concerns when administered intranasally. Sublingual immunization has received more attention in recent years based on its efficiency in inducing systemic and mucosal immune responses in both mucosal and extra-mucosal tissues. To facilitate the transport of the immunogen across the sub-mucosal epithelial barrier, we found that CD91, the receptor of C1q, is prevalently expressed in the sublingual mucosal lining, and thus, a modified chimeric C1q surface conjugated CD40L/HIV VLP was generated. The ability of this chimeric C1q/CD40L/HIV VLP to bind, cross the epithelial layer, access and activate the sub-mucosal layer dendritic cells (DCs), and ultimately induce enhanced mucosal and systemic immune responses against HIV is evaluated in this study. We found that C1q/CD40L/HIV VLPs have enhanced binding, increased transport across the epithelial layer, and upregulate DC activation markers as compared to CD40L/HIV VLPs alone. Mice immunized with C1q/ CD40L/HIV VLPs by sublingual administration showed higher levels of IgA salivary antibodies against both HIV Gag and Env than mice immunized with CD40L/HIV VLPs. Moreover, sublingual immunization with C1q/CD40L/HIV VLPs induced more Env- and Gag-specific IFN-γ producing T cells than the CD40L/HIV VLPs group. Interestingly, C1q/CD40L/HIV VLP immunization can also induce more mucosal homing T cells than that in CD40L/HIV VLP group. Our data suggest that incorporation of C1q to CD40L/HIV VLPs is a promising novel strategy and that the sublingual immunization can be a favorite immunization route for HIV mucosal vaccines.

A ROLE FOR CELLULAR IMMUNITY IN EARLY POSTPARTUM PERIOD

The functioning of the secretory organs is closely related to the activity of the immune system. As is well known, this participation is manifested in the fact that at certain stages of activity, the lymphoid cells migrating to the organ can be involved in the regulation of secretion. In addition, the products of the immune system and even its cellular elements can become components of a number of secrets. Colostrum and milk contain a large number of cells of a wide spectrum (up to 1/3 of the volume), of which the number of lymphocytes is up to 16% of leukocytes. Lymphocytes, in an immunologically active form, entering the newborn’s body with colostrum, activate the cellular immunity system. The transport of lymphokinin mediators plays a certain role in this process. Microphages, T- and B-lymphocytes, penetrating through the intercellular spaces into the lymphoid layer of the intestine, transmit immunoreceptors to the prolymphocytes of the newborn, "armed" with their activity to recognize genetically foreign ones. The lymphocytes contained in colostrum are the cells of the immune system that provide cellular and humoral immunity. They are mainly represented by T-cells, B-cells and killer cells. Milk T-cells produce a full spectrum of immune regulatory proteins such as interferon, tumor necrosis factor alpha. These cells are the cells of the immune memory. Newborns who received the first portion of colostrum no later than an hour after birth are characterized by an increased number of leukocytes, more pronounced phagocytosis, which indicates the stimulation of hemo- and lymphocytosis. When carrying out transmission and scanning electron microscopy in the epithelial layer of the intestine, cellular elements were found that got there from the intestinal lumen. Microsections show how cells of a lymphoid nature, pushing apart the structures of the epithelial layer, bypass natural barriers and, at the same time, retain their physiological usefulness. The possibility of penetration of immunocompetent cells of the mother’s colostrum into the bloodstream of the young is proved using the natural label of the female’s cells – sex chromatin. Naturally, sex chromatin-labeled cells were sought in male newborns. The detection of colostrum cells in the intestinal wall and bloodstream of the young is approximately 25% in the blood, 1% in the lymph, and about 70% in the intestine. There is no doubt that the leukocytes of colostrum are of exceptional importance in creating immunity in newborn animals.

Tumor-Cell Invasion Initiates at Invasion Hotspots, an Epithelial Tissue-Intrinsic Microenvironment

Malignant cancers emerge in epithelial tissues through a progressive process in which a single transformed mutant cell becomes tumorigenic and invasive. Although numerous genes involved in the malignant transformation of cancer cells have been described, how tumor cells launch an invasion into the basal side of epithelial tissues remains elusive. Here, using a Drosophila wing imaginal disc epithelia, we show that genetically mosaic clones of cells mutant for a neoplastic-tumor-suppressor gene (nTSG) in combination with the oncogenic Ras (RasV12) expression initiate invasion into the basal side of the epithelial layer at specific spots in the epithelial tissue. In this "invasion hotspot", the oncogenic double-mutant cells activate c-Jun N-terminal kinase (JNK) signaling, which causes basal extrusion of the double-mutant cells and destruction of basement membrane through upregulation of a matrix metalloprotease, MMP1. Conversely, in other regions of the epithelial tissue, the double-mutant cells do not strongly activate JNK, deviate from the apical side of the epithelial layer, and show benign tumor growth in the lumen. These data indicate that the onset of tumor-cell invasion is highly dependent on the tissue-intrinsic local microenvironment. Given the conservation of genetic signaling pathways involved in this process, initiation of tumor-cell invasion from invasion hotspots in Drosophila wing imaginal epithelia could help us to understand the developmental mechanisms of invasive cancers.

Apical contacts stemming from incomplete delamination guide progenitor cell allocation through a dragging mechanism

The developmental strategies used by progenitor cells to allow a safe journey from their induction place towards the site of terminal differentiation are still poorly understood. Here, we uncovered a mechanism of progenitor cell allocation that stems from an incomplete process of epithelial delamination that allows progenitors to coordinate their movement with adjacent extra-embryonic tissues. Progenitors of the zebrafish laterality organ originate from the superficial epithelial enveloping layer by an apical constriction process of cell delamination. During this process, progenitors retain long-lasting apical contacts that enable the epithelial layer to pull a subset of progenitors on their way to the vegetal pole. The remaining delaminated cells follow the movement of apically attached progenitors by a protrusion-dependent cell-cell contact mechanism, avoiding sequestration by the adjacent endoderm, ensuring their collective fate and allocation at the site of differentiation. Thus, we reveal that incomplete delamination serves as a cellular platform for coordinated tissue movements during development.

Transepithelial potential difference governs epithelial homeostasis by electromechanics

Studies of electric effects in biological systems, from the historical experiments of Galvani 1 and the ground-breaking work on action potential2 to studies on limb regeneration3 or wound healing4, share the common feature of being concerned with transitory behavior and not addressing the question of homeostasis. Here using a novel microfluidic device, we study how the homeostasis of confluent epithelial tissues is modified when a trans-epithelial electric potential (TEPD) different from the natural one is imposed on an epithelial layer. We show that epithelial fate is dependent on TEPD of few Volts/cm similar to the endogenous one. When the field direction matches the natural one, we can restore a perfect confluence in an epithelial layer turned defective either by E-cadherin knock-out or by weakening cell-substrate adhesion; additionally, the tissue pushes on the substrate with kilo-Pascals stress, inducing active cell response such as death and differentiation. When the field is opposite, homeostasis is destroyed by the perturbation of junctional actin and cell shapes, and the formation of dynamical mounds5, while the tissue pulls with similar strengths. Most of these observations can be quantitatively explained by an electro-hydrodynamic theory involving local electro-osmotic flows. We expect this work to motivate further studies on long time effects of electromechanical pathways with important tissue engineering applications.

Preparation of Single Cell Suspensions of the Intra-Epithelial Layer and Lamina Propria from Human Intestinal Tissue v1

This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from the epithelial layer and the lamina propria of human gut sections of about one gram of tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples. This protocol can be used for any section of the intestinal tract from duodenum to distal colon.

Adipose tissue versus stem cell-derived small extracellular vesicles to enhance the healing of acute burns

Aim: Proper healing of extensive burns remains a healthcare challenge. In the present study, we proposed a distinct therapeutic application of adipose tissue and small extracellular vesicles isolated from human menstrual blood-derived mesenchymal stem cells (MenSC) small extracellular vesicles (sEVs) to enhance the repair of third-degree burn injury. Materials & methods: Mouse model of third-degree burn was used. Adipose tissue in the form of Nano-fat (NF) and MenSC-sEVs was injected subcutaneously at the site of injuries. Results: NF and sEVs were capable of enhancing wound closure and increasing neoangiogenesis. NF was also effective in accelerating the formation of granulation tissue and boosting the thickness of the new epithelial layer. Conclusion: This study demonstrates the effectiveness of NF and MenSC-sEVs as promising therapeutic approaches to facilitate the repair of skin burns.

Contribution of Environmental Constituents in the Genomic Disruption of Cytokeratins

Cytokeratins are keratinous protein and assist cells to reduce mechanical stress on the intracytoplasmic layer of epithelial tissue. There are several unspecified mutations in the epithelial layer that may induces by environmental mutagens and pathogens. The unspecified mutations in the epithelium surface also disrupt biology of skin at multiple different levels and cause innate keratinizing disorders. These serve as a root generator of neurohormones and neuropeptides which mainly partake in the disruption. Generally, all 54 unique genes of human keratin partake in mutations and cause cutaneous tissue fragility, skin hypertrophic, and malignant transformation. In this chapter, unspecific factors that involved in the pathogenesis of skin diseases and the ways by which such keratin changes might harness to alleviate different skin conditions are also included. Consequently, the contribution of environmental changes in the frontier of mutations or misregulations of the cytokeratin genes, is also cited here.

RISK FACTORS FOR MALIGNANCY OF PAPILLOMAS IN THE OROPHARYNGEAL MUCOUS MEMBRANE ASSOCIATED WITH HUMAN PAPILLOMAVIRUS (HPV)

Analysis of the HPV positive papilloma structures at different levels of pathological process development was carried out. Classical staining of preparations with hematoxylin and eosin was used for immunohistochemical determination of Ki67-positive cells and phenotyping of CD positive cells. We found that the process of papilloma formation begins with a local increase in the proliferative activity of keratinocytes which contributes to the formation of a local epithelial convex above the surface of the oropharyngeal mucosa (OPM) in the form of papilloma. The connective tissue of the OPM adjacent to the epithelium grows at the second stage. At the same time here is a decrease in immunocytes/macrophages number of the oral epithelium not only in the growth zone of the papilloma but in the adjacent neoplasm tissue. The third stage is characterized by the destruction of the basal membrane of the OPM. Apoptotic cells in the cambial layer and forming leukocytes infiltrate the OPM lamina propria. Virus-infected keratinocytes are phagocytized by macrophages or exfoliated from the surface of the epithelial layer. The emerging in the middle layers defect of tissue of the epithelial layer and the absence of Langerhans cells indicate a relationship between the migration of antigen-presenting cells expressing CD68 with impaired differentiation and specialization of keratinocytes. The conclusion is based on the analogy of "leukemic failure" in leukemia and on the absence of differentiating epithelial cells between the cambium and the specialized surface layer in the long-existing papilloma. The disappearance from the epithelium of CD68-positive cells specific to the epithelial layer is a prognostic sign of malignization in the mucous membrane of the oropharynx.