Effects of Perfluorohexane Sulfonate Exposure on Immune Cell Populations in Naive Mice.

Perfluorohexane sulfonate (PFHxS) is a member of the per- and polyfluoroalkyls (PFAS) superfamily of molecules, characterized by their fluorinated carbon chains and use in a wide range of industrial applications. PFHxS and perfluorooctane sulfonate are able to accumulate in the environment and in humans with the approximated serum elimination half-life in the range of several years. More recently, some PFAS compounds have also been suggested as potential immunosuppressants. In this study, we analyze immune cell numbers in mice following 28-d repeated oral exposure to potassium PFHxS at 12, 120, 1,200, and 12,000 ng/kg/d, with resulting serum levels ranging up to ∼1,600 ng/ml, approximating ranges found in the general population and at higher levels in PFAS workers. The immunosuppressant cyclophosphamide was analyzed as a positive control. B cells, T cells, and granulocytes from the bone marrow, liver, spleen, lymph nodes, and thymus were evaluated. We found that at these exposures, there was no effect of PFHxS on major T or B cell populations, macrophages, dendritic cells, basophils, mast cells, eosinophils, neutrophils, or circulating Ab isotypes. By contrast, mice exposed to cyclophosphamide exhibited depletion of several granulocyte and T and B cell populations in the thymus, bone marrow, and spleen, as well as reductions in IgG1, IgG2b, IgG2c, IgG3, IgE, and IgM. These data indicate that exposures of up to 12,000 ng/kg of PFHxS for 28 d do not affect immune cell numbers in naive mice, which provides valuable information for assessing the risks and health influences of exposures to this compound.

[Homing and characteristic analysis of macrophage in immune-mediated aplastic anemia model mice].

To investigate the dynamic homing process and characteristics of macrophages in different organs of immune-mediated aplastic anemia (AA) model mice. Macrophages in donor lymph nodes were sorted by magnetic beads and labeled with PKH67. After modeling according to the preparation method of the AA model, peripheral blood rountine analysis, bone marrow biopsy and HE staining results were analyzed to verify the modeling effect. On days 4, 8, and 12 of modeling, the bone marrow, spleen, and lymph node mononuclear cells were collected, and dynamic changes of PKH67-labeled macrophages in donor mice were analyzed by flow cytometry. In this study, dynamic changes in PKH67-labeled macrophages in the pathogenesis of AA model mice were explored. Macrophages in donor mice homed to the lymph nodes, expanding and differentiating in the lymph nodes, and finally transported to the bone marrow and spleen. Through proteomics mass spectrometry analysis, the related immune inflammatory response pathway of macrophages involved in the activation of the AA bone marrow microenvironment was preliminarily revealed, which provides a basis for the pathological macrophages involved in the pathogenesis of AA model mice.

Immunostimulant effect of dates (Phoenix dactylifera) on humoral and cellular immunity cells and their functions.

The study aimed to assess the immunomodulatory effects of Phoenix dactylifera (dates) fruit, a traditional remedy used by Moroccans to enhance immunity against pathogens. This research sought to evaluate the impacts of this fruit on immune cells and their functions. To achieve this, we conducted tests using date extracts on splenocytes, thymocytes, and macrophages, focusing on their functions: antibody production, phagocytosis, and T-lymphocyte toxicity. The results obtained demonstrated that the aqueous extract of P. dactylifera fruit exhibited significant immunostimulatory effects on humoral immunity. It achieved this by enhancing complement activity and increasing splenocyte (including B-lymphocytes) proliferation by 142.5% compared to control cells. Similarly, in the same conditions, there was notable stimulation of cellular immunity through thymocyte activity, resulting in a remarkable increase in cell proliferation (225%) and a boost in thymocyte function (245.9%), which plays a role in safeguarding against cancer. Moreover, the date extract demonstrated anti-inflammatory properties. This was evident in the increased phagocytosis activity mediated by macrophages under the ethyl acetate extract, effectively eliminating pathogens. Assessing the cosmetic potential of date extracts showed that the ethyl acetate extract possesses both anti-inflammatory and strong antioxidant effects, exhibited high photo absorption of ultraviolet-B rays. Based on these findings, we propose to study the utilization of this extract for sun protection as a sunscreen. Furthermore, the Fourier-transform infrared spectroscopy analysis indicated that the most active compounds present were flavonoids. These outcomes substantiate the traditional usage of this fruit for reinforcing immunity.

15-epi-lipoxin A5 promotes neutrophil exit from exudates for clearance by splenic macrophages.

Specialized proresolving mediators (SPMs) promote local macrophage efferocytosis but excess leukocytes early in inflammation require additional leukocyte clearance mechanism for resolution. Here, neutrophil clearance mechanisms from localized acute inflammation were investigated in mouse dorsal air pouches. 15-HEPE (15-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid) levels were increased in the exudates. Activated human neutrophils converted 15-HEPE to lipoxin A5 (5S,6R,15S-trihydroxy-7E,9E,11Z,13E,17Z-eicosapentaenoic acid), 15-epi-lipoxin A5 (5S,6R,15R-trihydroxy-7E,9E,11Z,13E,17Z-eicosapentaenoic acid), and resolvin E4 (RvE4; 5S,15S-dihydroxy-6E,8Z,11Z,13E,17Z-eicosapentaenoic acid). Exogenous 15-epi-lipoxin A5, 15-epi-lipoxin A4 and a structural lipoxin mimetic significantly decreased exudate neutrophils and increased local tissue macrophage efferocytosis, with comparison to naproxen. 15-epi-lipoxin A5 also cleared exudate neutrophils faster than the apparent local capacity for stimulated macrophage efferocytosis, so the fate of exudate neutrophils was tracked with CD45.1 variant neutrophils. 15-epi-lipoxin A5 augmented the exit of adoptively transferred neutrophils from the pouch exudate to the spleen, and significantly increased splenic SIRPa+ and MARCO+ macrophage efferocytosis. Together, these findings demonstrate new systemic resolution mechanisms for 15-epi-lipoxin A5 and RvE4 in localized tissue inflammation, which distally engage the spleen to activate macrophage efferocytosis for the clearance of tissue exudate neutrophils.

Basement Membrane Matrix Encapsulated Cell Aggregation for Investigating Murine Spleen Tissue Formation.

The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases susceptibility to severe blood infections and sepsis. Auto-transplantation of spleen slices has been used clinically to replace lost tissue and restore immune function. However, the mechanism driving robust and immunologically functional spleen tissue regeneration has not been fully elucidated. Here, we aim to develop a method for aggregating and encapsulating spleen cells within a semi-solid matrix in order to investigate the cellular requirements for spleen tissue formation. Basement membrane matrix encapsulated cell constructs are amenable to both in vitro tissue culture of three-dimensional organoids as well as transplantation under the kidney capsule to directly assess in vivo tissue formation. By manipulating the input cells for aggregation and encapsulation, we demonstrate that graft-derived PDGFRß+MAdCAM-1- neonatal stromal cells are required for spleen tissue regeneration under animal transplantation models.

Inhibition of the p62-Nrf2-GPX4 Pathway Confers Sensitivity to Butachlor-Induced Splenic Macrophage Ferroptosis.

Butachlor is widely used in agriculture around the world and therefore poses environmental and public health hazards due to persistent and poor biodegradability. Ferroptosis is a type of iron-mediated cell death controlled by glutathione (GSH) and GPX4 inhibition. P62 is an essential autophagy adaptor that regulates Keap1 to activate nuclear factor erythroid 2-related factor 2 (Nrf2), which effectively suppresses lipid peroxidation, thereby relieving ferroptosis. Here, we found that butachlor caused changes in splenic macrophage structure, especially impaired mitochondrial morphology with disordered structure, which is suggestive of the occurrence of ferroptosis. This was further confirmed by the detection of iron metabolism, the GSH system, and lipid peroxidation. Mechanistically, butachlor suppressed the protein level of p62 and promoted Keap1-mediated degradation of Nrf2, which results in decreased GPX4 expression and accelerated splenic macrophage ferroptosis. These findings suggest that targeting the p62-Nrf2-GPX4 signaling axis may be a promising strategy for treating inflammatory diseases.

In vitro antiviral activity of eugenol on Singapore grouper iridovirus.

The high mortality rate of Singapore grouper iridovirus (SGIV) posing a serious threat to the grouper aquaculture industry and causing significant economic losses. Therefore, finding effective drugs against SGIV is of great significance. Eugenol (C10H12O2) is a phenolic aromatic compound, has been widely studied for its anti-inflammatory, antioxidant and antiviral capacity. In this study, we explored the effect of eugenol on SGIV infection and its possible mechanisms using grouper spleen cells (GS) as an in vitro model. We found that treatment of GS cells with 100 µM eugenol for 4 h exhibited the optimal inhibitory effect on SGIV. Eugenol was able to reduce the expression level of inflammatory factors by inhibiting the activation of MAPK pathway and also inhibited the activity of NF-κB and AP-1 promoter. On the other hand, eugenol attenuated cellular oxidative stress by reducing intracellular ROS and promoted the expression of interferon-related genes. Therefore, we conclude that eugenol inhibits SGIV infection by enhancing cellular immunity through its anti-inflammatory and antioxidant functions.

Celecoxib exhibits antifungal effect against Paracoccidioides brasiliensis both directly and indirectly by activating neutrophil responses.

BACKGROUND: Celecoxib, an anti-inflammatory drug, combined therapies using antimicrobials and immune modulator drugs are being studied. OBJECTIVE: To assess whether Celecoxib has direct in vitro antifungal effect against the Paracoccidioides brasiliensis, the causative agent of Paracoccidioidomycosis-(PCM) and also if it improves the in vivo activity of neutrophils-(PMN) in an experimental murine subcutaneous-(air pouch) model of the disease. METHODS: The antifungal activity of Celecoxib(6 mg/mL) on P. brasiliensis-(Pb18) was evaluated using the microdilution technique. Splenocytes co-cultured with Pb18 and treated with Celecoxib(6 mg/mL) were co-cultured for 24, 48 and 72-hours. Swiss mice were inoculated with Pb18 and treated with Celecoxib(6 mg/kg) in the subcutaneous air pouch. Neutrophils were collected from the air pouch. Mitochondrial activity, reactive oxygen production, catalase, peroxidase, cytokines and chemokines, nitrogen species, total protein, microbicidal activity of PMNs and viable Pb18 cells numbers were analyzed. RESULTS: Celecoxib had no cytotoxic effect on splenocytes co-cultured with Pb18, but had a marked direct antifungal effect, inhibiting fungal growth both in vitro and in vivo. Celecoxib interaction with immune system cells in the air pouch, it leads to activation of PMNs, as confirmed by several parameters (mitochondrial activity, reactive oxygen species, peroxidase, KC and IL-6 increase, killing constant and phagocytosis). Celecoxib was able to reduce IL-4, IL-10 and IL-12 cytokine production. The number of recovered viable Pb18 decreased dramatically. CONCLUSIONS: This is the first report of the direct antifungal activity of Celecoxib against P. brasiliensis. The use of Celecoxib opens a new possibility for future treatment of PCM.

Immunostimulating effects of ulvan type polysaccharide isolated from Korean Ulva pertusa in cyclophosphamide-induced immunosuppressed BALB/c mice.

This study aimed to determine the immunostimulatory activities of ulvan type polysaccharides isolated from Ulva pertusa. First, U. pertusa polysaccharide (UPP) mainly consists of rhamnose, glucuronic acid, iduronic acid, and xylose, which are typical ulvan type monosaccharides. UPP induced phosphorylation of the mitogen-activated protein kinase and nuclear factor-kappa B pathways in macrophages, subsequently triggering cytokine release and phagocytosis. The effects were closely associated with pattern recognition receptors such as dectin-1, mannose receptor, CD11b, CD14, and Toll-like receptors 2 and 4. Moreover, prophylactic administration of UPP was found to protect against body weight loss and lymphatic organ damage in cyclophosphamide-induced immunosuppressed mice. In addition, UPP demonstrated significant stimulatory effects on various immunocytes, such as T cells, B cells, macrophages, and natural killer cells derived from the spleen. These effects were closely related to the mitogen-activated protein kinase and nuclear factor-kappa B pathways, and significant secretion of immunostimulatory cytokines such as IL-6, -12, and TNF-α was noted in both blood and spleen samples. Impairment of the short-chain fatty acid balance in the cecum was prevented by UPP administration in a dose-dependent manner. Consequently, these results suggest that the UPP isolated from U. pertusa contributes to immune system activation.

Insights into microstructure and expression of markers of proliferation, apoptosis and T cells in the spleen of cattle egret (Bubulcus ibis).

The spleen is the largest secondary lymphoid organ with significant roles in pathogen clearance. It is involved in several avian diseases. The cattle egret is a wild insectivorous bird of agricultural and socioeconomic importance. Data related to microstructural features of cattle egret spleen are lacking. The present study investigated the gross anatomical, histological and immunohistochemical characteristics of the cattle egret spleen. Proliferation (PCNA and PHH3), apoptosis (cleaved caspase 3, C.CASP3) and T-cell (CD3 and CD8) markers were assessed. Grossly, the spleen appeared brownish red, oval-shaped and located at the oesophago-proventricular junction. Histologically, the spleen was surrounded by a thin capsule sending a number of trabeculae which contained branches of the splenic vessels. The white pulp consisted of the periarteriolar lymphoid sheath and periellipsoidal lymphatic sheath (PELS). The red pulp was formed of sinusoids and cords. The penicillar capillaries, which represent the terminal segments of the splenic arterial tree were highly branched, wrapped by prominent ellipsoids and directly connected to the splenic sinusoids, suggesting a closed type of circulation. Immunohistochemically, proliferating cell nuclear antigen (PCNA)-expressing cells were distributed with high counts throughout the splenic parenchyma, being highest within the splenic cords and PELS. Both PHH3- and C.CASP3-expressing cells revealed a similar pattern to that of PCNA, although with fewer counts. Large numbers of T cells were observed throughout the splenic parenchyma, mainly within the cords, as revealed by CD3 and CD8 immunoreaction. The present study provides a clear insight into the precise structure of the spleen in cattle egrets and thus improves our understanding about birds' immunity.

Transcriptomic diversity of innate lymphoid cells in human lymph nodes compared to BM and spleen.

Innate lymphoid cells (ILCs) are largely tissue-resident, mostly described within the mucosal tissues. However, their presence and functions in the human draining lymph nodes (LNs) are unknown. Our study unravels the tissue-specific transcriptional profiles of 47,287 CD127+ ILCs within the human abdominal and thoracic LNs. LNs contain a higher frequency of CD127+ ILCs than in BM or spleen. We define independent stages of ILC development, including EILP and pILC in the BM. These progenitors exist in LNs in addition to naïve ILCs (nILCs) that can differentiate into mature ILCs. We define three ILC1 and four ILC3 sub-clusters in the LNs. ILC1 and ILC3 subsets have clusters with high heat shock protein-encoding genes. We identify previously unrecognized regulons, including the BACH2 family for ILC1 and the ATF family for ILC3. Our study is the comprehensive characterization of ILCs in LNs, providing an in-depth understanding of ILC-mediated immunity in humans.

Shiikuwasha leaf and peel extracts inhibit allergic reactions by suppressing degranulation in RBL-2H3 rat basophilic leukemia cells and immunoglobulin production in mouse spleen lymphocytes.

This study aims to investigate the antiallergic effects of Shiikuwasha (Citrus depressa Hayata) leaf and peel extracts by examining the regulation of degranulation and inflammatory cytokine production from rat basophilic leukemia (RBL-2H3) cells and antigen-specific antibody production in sensitized mouse spleen lymphocytes. In vivo antiallergic activity was evaluated using the passive cutaneous anaphylaxis (PCA) reaction model. Extracts of Shiikuwasha leaves and peel were prepared using 80% methanol and dissolved in dimethylsulfoxide. The dinitrophenyl-human serum albumin-induced ß-hexosaminidase levels in immunoglobulin (Ig) E-sensitized RBL-2H3 cells were assessed using enzymatic assays. Cytokine production was measured by enzyme-linked immunosorbent assay. Antibody production capacity was evaluated using lymphocytes isolated from spleens of type I allergy model mice. Lymphocytes were cultured for 72 h with Shiikuwasha extracts, and ovalbumin-specific IgE, IgG1, and IgG2a levels were measured. Shiikuwasha leaf and peel extract significantly reduced ß-hexosaminidase release and suppressed interleukin-4 and tumor necrosis factor-α production from RBL-2H3 cells. Ovalbumin-specific IgE and IgG1 production decreased in Shiikuwasha extract-treated lymphocytes. These extracts also significantly suppressed the PCA reaction. Shiikuwasha leaf and peel extract reduce degranulation in RBL-2H3 cells and antibody production in spleen-derived lymphocytes and therefore exhibit antiallergic effects.

Blockade of CD80/CD86-CD28 co-stimulation augments the inhibitory function of peptide antigen-specific regulatory T cells.

Mixed lymphocyte culture under the blockade of CD80/CD86-CD28 co-stimulation induces anergic (completely hyporesponsive) T cells with immune suppressive function (inducible suppressing T cells: iTS cells). Previously, iTS cell therapy has demonstrated outstanding benefits in clinical trials for organ transplantation. Here, we examined whether peptide antigen-specific iTS cells are inducible. DO 11.10 iTS cells were obtained from splenocytes of BALB/c DO 11.10 mice by stimulation with OVA peptide and antagonistic anti-CD80/CD86 mAbs. When DO 11.10 iTS or Foxp3- DO 11.10 iTS cells were stimulated with OVA, these cells produced IL-13, but not IL-4. DO 11.10 iTS cells decreased IL-4 and increased IL-13 production from OVA-stimulated naïve DO 11.10 splenocytes. When Foxp3+ DO 11.10 iTS cells were prepared, these cells significantly inhibited the production of IL-4 and IL-13 compared with freshly isolated Foxp3+ DO 11.10 T cells. Moreover, an increase in the population expressing OX40, ICOS, and 4-1BB suggested activation of Foxp3+ DO 11.10 iTS cells. Thus, blockade of CD80/CD86-CD28 co-stimulation during peptide antigen stimulation augments the inhibitory function of Foxp3+ regulatory T cells, and does not induce anergic Foxp3- conventional T cells. Peptide-specific Foxp3+ regulatory iTS cells could be useful for the treatment of allergic and autoimmune diseases without adverse effects.

IL-10 Neutralization Attenuates Mast Cell Responses in a Murine Model of Experimental Food Allergy.

IgE-mediated mast cell (MC) activation is a critical component of allergic responses to oral Ags. Several T cell-derived cytokines have been shown to promote MC reactivity, and we recently demonstrated a critical role for the cytokine IL-10 in mediating MC responses during food allergy. In this study, we further validate the role of IL-10 using Ab-mediated IL-10 depletion. IL-10 neutralization significantly attenuated MC responses, leading to decreased MC accumulation and activation, as well as inhibition of MC-mediated symptoms such as allergic diarrhea. This was accompanied by decreased Th2 cytokine gene expression, attenuated systemic T cell responses, and fewer CD4 T cells, B cells, and MCs in the spleen. Our data further confirm the role of IL-10 in driving MC responses and suggest that IL-10-responsive MCs may constitute an important player in allergic responses.

Singapore grouper iridovirus VP146 modulates the cGAS-STING signaling pathway to escape the interferon immune response.

Singapore grouper iridovirus (SGIV) is a large double-stranded DNA virus that has caused significant economic losses to the grouper aquaculture industry. So far, the structure and function of SGIV proteins have been successively reported. In the present paper, the protein of SGIV VP146 was cloned and identified. VP146 was whole-cell distributed in GS cells. VP146 promoted SGIV replication and inhibited the transcription of interferon-related genes as well as pro-inflammatory cytokines in GS cells. In addition, VP146 was involved in the regulation of the cGAS-STING signaling pathway, and decreased cGAS-STING induced the promoter of ISRE and NF-κB. VP146 interacted with the proteins of cGAS, STING, TBK1, and IRF3 from grouper, but did not affect the binding of grouper STING to grouper TBK1 and grouper IRF3. Interestingly, grouper STING was able to affect the intracellular localization of VP146. Four segment structural domains of grouper STING were constructed, and grouper STING-CTT could affect the intracellular localization of VP146. VP146 had no effect on the self-binding of EcSITNG, nor on the binding of EcSTING to EcTBK1 and EcIRF3. Together, the results demonstrated that SGIV VP146 modulated the cGAS-STING signaling pathway to escape the interferon immune response.

Streptococcal pyrogenic exotoxin B is a superantigen that induces murine splenocyte proliferation and secretion of IL-2 and IFN-γ ex vivo.

Streptococcus pyogenes is a significant human pathogen, producing a range of virulence factors, including streptococcal pyrogenic exotoxin B (SpeB) that is associated with foodborne outbreaks. It was only known that this cysteine protease mediates cleavage of transmembrane proteins to permit bacterial penetration and is found in 25% of clinical isolates from streptococcal toxic shock syndrome patients with extreme inflammation. Its interaction with host and streptococcal proteins has been well characterized, but doubt remains about whether it constitutes a superantigen. In this study, for the first time it is shown that SpeB acts as a superantigen, similarly to other known superantigens such as staphylococcal enterotoxin A or streptococcal pyrogenic exotoxin type C, by inducing proliferation of murine splenocytes and cytokine secretion, primarily of interleukin-2 (IL-2), as shown by cytometric bead array analysis. IL-2 secretion was confirmed by enzyme-linked immunosorbent assay (ELISA) as well as secretion of interferon-γ. ELISA showed a dose-dependent relationship between SpeB concentration in splenocyte cells and IL-2 secretion levels, and it was shown that SpeB retains activity in milk pasteurized for 30 min at 63°C.

ß-Glucan Subverts the Function of Myeloid Cells in Neonates.

ß-Glucan is the main component of the cell wall of pathogen-associated molecular patterns (PAMPs) including various yeast, fungi, or certain bacteria. Previous reports demonstrated that ß-glucan was widely investigated as a potent immunomodulators to stimulate innate and adaptive immune responses, which indicated that it could be recommended as an effective adjuvant in immunotherapy. However, the detailed effects of ß-glucan on neonatal immunity are still largely unknown. Here, we found that ß-glucan did not affect the frequencies and numbers of myeloid cells in the spleen and bone marrow from neonates. Functional assay revealed that ß-glucan from neonates compromised the immunosuppressive function of immature myeloid cells, which were myeloid-derived suppressor cells (MDSCs). Flow cytometry or gene expression analysis revealed that ß-glucan-derived polymorphonuclear (PMN)-MDSCs produced lower level of reactive oxygen species (ROS) and arginase-1 (Arg1) in neonatal mice. Furthermore, ß-glucan administration significantly decreased the frequency and ROS level of PMN-MDSCs in vitro. These observations suggest that ß-glucan facilitates the maturation of myeloid cells in early life, which may contribute to its beneficial effects against immune disorders later in life.

Structure characterization and intestinal immune promotion effect of polysaccharide purified from Alhagi camelorum Fisch.

This study investigated the structure of acid Alhagi camelorum Fischa polysaccharide (aAP) and its impact on intestinal activity in mice. The results showed that aAP comprised of the fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid with the molar ratio of 0.81:14.97:10.84:11.14:3.26:0.80:0.80:54.92:2.47 with the molecular weight (Mw) of 22.734 kDa. Additionally, the composition of aAP was assessed via FT-IR, methylation, and NMR analyses, indicating that the backbone of the aAP was consisted of →4)-α-D-GalpA-6-OMe-(1 â†’ 4)-α-GalpA-(1 â†’ and →4)-α-D-GalpA-6-OMe-(1 â†’ 2)-α-L-Rhap-(1→, as well as →4)-ß-D-Galp- and →5)-α-L-Araf- for the branched chain. Furthermore, ICR mice underwent intragastric administration of different concentrations of aAP for 7 consecutive days. The results showed that aAP enhanced the murine spleen and thymus indices, promoted the secretion of serum lgG antibody, intestinal lgA antibody and intestinal cytokines, improved the morphology of intestinal villi and crypts, enhanced quantity of intestinal IELs and IgA+ cells, and activated T lymphocytes and DC cells in MLNs. In summary, these findings suggest that the utilization of aAP could enhance the immune response of the murine intestinal mucosa.

Immunomodulatory properties of Bacillus subtilis extracellular vesicles on rainbow trout intestinal cells and splenic leukocytes.

Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that carry bioactive molecules. Among EVs, outer membrane vesicles (OMVs), specifically produced by Gram-negative bacteria, have been extensively characterized and their potential as vaccines, adjuvants or immunotherapeutic agents, broadly explored in mammals. Nonetheless, Gram-positive bacteria can also produce bilayered spherical structures from 20 to 400 nm involved in pathogenesis, antibiotic resistance, nutrient uptake and nucleic acid transfer. However, information regarding their immunomodulatory potential is very scarce, both in mammals and fish. In the current study, we have produced EVs from the Gram-positive probiotic Bacillus subtilis and evaluated their immunomodulatory capacities using a rainbow trout intestinal epithelial cell line (RTgutGC) and splenic leukocytes. B. subtilis EVs significantly up-regulated the transcription of several pro-inflammatory and antimicrobial genes in both RTgutGC cells and splenocytes, while also up-regulating many genes associated with B cell differentiation in the later. In concordance, B. subtilis EVs increased the number of IgM-secreting cells in splenocyte cultures, while at the same time increased the MHC II surface levels and antigen-processing capacities of splenic IgM+ B cells. Interestingly, some of these experiments were repeated comparing the effects of B. subtilis EVs to EVs obtained from another Bacillus species, Bacillus megaterium, identifying important differences. The data presented provides evidence of the immunomodulatory capacities of Gram-positive EVs, pointing to the potential of B. subtilis EVs as adjuvants or immunostimulants for aquaculture.

Optimization of methods for intrasplenic administration of human amniotic epithelial cells in order to perform safe and effective cell-based therapy for liver diseases.

In animal experimental models the administration of stem cells into the spleen should ensure high effectiveness of their implantation in the liver due to a direct vascular connection between the two organs. The aim of this study was to update the methods of experimental intrasplenic cell transplantation using human amniotic epithelial cells (hAECs) which are promising cells in the treatment of liver diseases. BALB/c mice were administered intrasplenically with 0.5, 1, and 2 million hAECs by direct bolus injection (400 µl/min) and via a subcutaneous splenic port by fast (20 µl/min) and slow (10 µl/min) infusion. The port was prepared by translocating the spleen to the skin pocket. The spleen, liver, and lungs were collected at 3 h, 6 h, and 24 h after the administration of cells. The distribution of hAECs, histopathological changes in the organs, complete blood count, and biochemical markers of liver damage were assessed. It has been shown that the method of intrasplenic cell administration affects the degree of liver damage. The largest number of mice showing significant liver damage was observed after direct administration and the lowest after slow administration through a port. Liver damage increased with the number of administered cells, which, paradoxically, resulted in increased liver colonization efficiency. It was concluded that the administration of 1 × 106 hAECs by slow infusion via a subcutaneous splenic port reduces the incidence of complications at the expense of a slight decrease in the effectiveness of implantation of the transplanted cells in the liver.