Mouse Lymphocytes Research Articles

V-domain immunoglobulin suppressor of T-cell activationand programmed death receptor 1 dual checkpoint blockade enhances antitumour immunity and survival in glioblastoma.

The current therapy cannot meet the needs of glioblastoma (GBM). V-domain immunoglobulin suppressor of T-cell activation (VISTA) is significantly up-regulated in GBM patients; however, its therapeutic potential in GBM is still unclear. Flow cytometry was used to detect the expression of VISTA and the co-expression pattern of VISTA and programmed death receptor 1 (PD-1) on brain infiltrating lymphocytes of GBM mice. Monoclonal antibody therapy was used to evaluate the therapeutic effect of α-VISTA monotherapy and α-VISTA combined with α-PD-1 on GBM mice. Transcriptome analysis, flow cytometry, and immunofluorescence were used to detect changes of immune microenvironment in mouse brain tumours. Immunofluorescence and TCGA data analysis were used to further validate the combined treatment strategy on patient data. Compared with normal mice, the frequency of VISTA expression and co-expression of VISTA and PD-1 on tumour-infiltrating lymphocytes (TILs) in tumour-bearing mice was increased. Anti-VISTA monotherapy significantly up-regulated multiple immune stimulation-related pathways and moderately prolonged mouse survival time. Blocking the immune checkpoint VISTA and PD-1 significantly prolonged the survival time of mice and cured about 80% of the mice; CD8+ T cells played an important role in this process. In addition, we found that the expression of VISTA and PD-1 was significantly up-regulated in GBM patients by immunofluorescence, and patients with high expression of VISTA and PD-1 were associated with poor overall survival. This combination of blocking the immune checkpoint VISTA and PD-1 may achieve clinical transformation in GBM.

Isoliquiritigenin Suppresses Breast Tumor Development by Enhancing Host Antitumor Immunity.

Isoliquiritigen (ISL), a constituent of licorice, has been shown to possess antitumorigenic effects in diverse cancer types. In this study, we observed that ISL suppressed breast tumor development significantly more effectively in immunocompetent mice than in immunocompromised ones. In exploring the cause of such a discrepancy, we detected robust tumor infiltration of CD8[Formula: see text] T lymphocytes in mice treated with ISL, not seen in tumors derived from vehicle-treated mice. Moreover, we found a dramatic reduction in PD-L1 in both experimental breast tumors and cultured breast cancer cells upon ISL treatment. In further experiments, we showed that ISL selectively elevated miR-200c in breast cancer and confirmed that PD-L1 mRNA is the target of miR-200c in both murine and human breast cancer cells. ISL suppression of PD-L1 was functionally linked to miR-200c/ZEB1/2 because (1) ISL diminished ZEB1/2; (2) knockdown of ZEB1/2 led to the disappearance of PD-L1; and (3) miR-200c antagomiR disabled ISL to reduce PD-L1. We found evidence that ISL reduced the level of PD-L1 by simultaneously intercepting the ERK and Src signaling pathways. In agreement with clinical finding that PD-L1 antibodies enhance efficacy of taxane-based therapy, we showed that ISL improved the tumoricidal effects of paclitaxel in an orthopedic murine breast tumor model. This study demonstrates that ISL-led tumor suppression acts through the augmentation of host antitumor immunity.

Astrocyte-induced Cdk5 expedites breast cancer brain metastasis by suppressing MHC-I expression to evade immune recognition.

Brain metastases (BrMs) evade the immune response to develop in the brain, yet the mechanisms of BrM immune evasion remains unclear. This study shows that brain astrocytes induce the overexpression of neuronal-specific cyclin-dependent kinase 5 (Cdk5) in breast cancer-derived BrMs, which facilitates BrM outgrowth in mice. Cdk5-overexpressing BrMs exhibit reduced expression and function of the class I major histocompatibility complex (MHC-I) and antigen-presentation pathway, which are restored by inhibiting Cdk5 genetically or pharmacologically, as evidenced by single-cell RNA sequencing and functional studies. Mechanistically, Cdk5 suppresses MHC-I expression on the cancer cell membrane through the Irf2bp1-Stat1-importin α-Nlrc5 pathway, enabling BrMs to avoid recognition by T cells. Treatment with roscovitine-a clinically applicable Cdk5 inhibitor-alone or combined with immune checkpoint inhibitors, significantly reduces BrM burden and increases tumour-infiltrating functional CD8+ lymphocytes in mice. Thus, astrocyte-induced Cdk5 overexpression endorses BrM immune evasion, whereas therapeutically targeting Cdk5 markedly improves the efficacy of immune checkpoint inhibitors and inhibits BrM growth.

Understanding the Role of miR-29a in the Regulation of RAG1, a Gene Associated with the Development of the Immune System.

The process of Ag receptor diversity is initiated by RAGs consisting of RAG1 and RAG2 in developing lymphocytes. Besides its role as a sequence-specific nuclease during V(D)J recombination, RAGs can also act as a structure-specific nuclease leading to genome instability. Thus, regulation of RAG expression is essential to maintaining genome stability. Previously, the role of miR29c in the regulation of RAG1 was identified. In this article, we report the regulation of RAG1 by miR-29a in the lymphocytes of both mice (Mus musculus) and humans (Homo sapiens). The level of RAG1 could be modulated by overexpression of miR-29a and inhibition using anti-miRs. Argonaute2-immunoprecipitation and high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation studies established the association of miR-29a and RAG1 with Argonaute proteins. We observed a negative correlation between miR-29a and RAG1 levels in mouse B and T cells and leukemia patients. Overexpression of pre-miR-29a in the bone marrow cells of mice led to the generation of mature miR-29a transcripts and reduced RAG1 expression, which led to a significant reduction in V(D)J recombination in pro-B cells. Importantly, our studies are consistent with the phenotype reported in miR-29a knockout mice, which showed impaired immunity and survival defects. Finally, we show that although both miR-29c and miR-29a can regulate RAG1 at mRNA and protein levels, miR-29a substantially impacts immunity and survival. Our results reveal that the repression of RAG1 activity by miR-29a in B cells of mice and humans is essential to maintain Ig diversity and prevent hematological malignancies resulting from aberrant RAG1 expression in lymphocytes.

Luteolin exerts anti-tumour immunity in hepatocellular carcinoma by accelerating CD8+ T lymphocyte infiltration.

Luteolin, a commonly used traditional Chinese medicine, has been utilized for several decades in the treatment of hepatocellular carcinoma (HCC). Previous research has demonstrated its anti-tumour efficacy, but its underlying mechanism remains unclear. This study aimed to assess the therapeutic effects of luteolin in H22 tumour-bearing mice. luteolin effectively inhibited the growth of solid tumours in a well-established mouse model of HCC. High-throughput sequencing revealed that luteolin treatment could enhance T-cell activation, cell chemotaxis and cytokine production. In addition, luteolin helped sustain a high ratio of CD8+ T lymphocytes in the spleen, peripheral blood and tumour tissues. The effects of luteolin on the phenotypic and functional changes in tumour-infiltrating CD8+ T lymphocytes were also investigated. Luteolin restored the cytotoxicity of tumour-infiltrating CD8+ T lymphocytes in H22 tumour-bearing mice. The CD8+ T lymphocytes exhibited intensified phenotype activation and increased production of granzyme B, IFN-γ and TNF-α in serum. The combined administration of luteolin and the PD-1 inhibitor enhanced the anti-tumour effects in H22 tumour-bearing mice. Luteolin could exert an anti-tumour immune response by inducing CD8+ T lymphocyte infiltration and enhance the anti-tumour effects of the PD-1 inhibitor on H22 tumour-bearing mice.

Effect of Collagen Peptides on Function of Mouse T Lymphocytes under Simulated Microgravity

To understand the effect of collagen peptides on the function of mouse lymphocytes under simulated microgravity. The splenocytes of mice were isolated, and the rotary cell culture system was used to simulate the microgravity. The T lymphocytes were stimulated with mitotic agents, concanavalin A (ConA), and the cells were treated with different concentrations of collagen peptides. The proliferation of lymphocytes and the levels of cytokines in the supernatant were detected. Simulated microgravity could inhibit the proliferation of spleen T lymphocytes and decrease the level of cytokines in the supernatant. Collagen peptides could promote the lymphocyte proliferation and cytokine production in cells cultured under simulated microgravity. Collagen peptides may attenuate the inhibitory effect of simulated microgravity on T lymphocytes by regulating the cell proliferation and the secretion of cytokines.

Enhancing Immune Responses against SARS-CoV-2 Variants in Aged Mice with INDUK: A Chimeric DNA Vaccine Encoding the Spike S1-TM Subunits.

Currently available vaccines against COVID-19 showed high efficacy against the original strain of SARS-CoV-2 but progressively lower efficacy against new variants. In response to emerging SARS-CoV-2 strains, we propose chimeric DNA vaccines encoding the spike antigen, including a combination of selected key mutations from different variants of concern. We developed two DNA vaccines, pVAX-S1-TM-D614G and pVAX-S1-TM-INDUK (INDUK), encoding the SARS-CoV-2 S1 spike subunit in fusion with the transmembrane region that allows protein trimerization as predicted by in silico analysis. pVAX-S1-TM-D614G included the dominant D614G substitution, while the chimeric vaccine INDUK contained additional selected mutations from the Delta (E484Q and L452R) and Alpha (N501Y and A570D) variants. Considering that aging is a risk factor for severe disease and that suboptimal vaccine responses were observed in older individuals, the immunogenicity of pVAX-S1-TM-D614G and INDUK was tested in both young and aged C57BL/6 mice. Two vaccine doses were able to trigger significant anti-SARS-CoV-2 antibody production, showing neutralizing activity. ELISA tests confirmed that antibodies induced by pVAX-S1-TM-D614G and INDUK were able to recognize both Wuhan Spike and Delta variant Spike as trimers, while neutralizing antibodies were detected by an ACE2:SARS-CoV-2 Spike S1 inhibitor screening assay, designed to assess the capacity of antibodies to block the interaction between the viral spike S1 protein and the ACE2 receptor. Although antibody titer declined within six months, a third booster dose significantly increased the magnitude of humoral response, even in aged individuals, suggesting that immune recall can improve antibody response durability. The analysis of cellular responses demonstrated that vaccination with INDUK elicited an increase in the percentage of SARS-CoV-2-specific IFN-γ producing T lymphocytes in immunized young mice and TNF-α-producing T lymphocytes in both young and aged mice. These findings not only hold immediate promise for addressing evolving challenges in SARS-CoV-2 vaccination but also open avenues to refine strategies and elevate the effectiveness of next-generation vaccines.

Anti-tumoral Immunity and Chemo-preventive Effectiveness of Herbal Extracts of Curcumin, Ginger, Clove and Amygdaline in Ehrlich Ascites Carcinoma-Challenging Mice.

Due to its systemic toxicity, traditional chemotherapy of tumors is being taken into consideration. Herbal therapy, containing phytochemical polyphenol derivatives such as Curcumin (Cur), Ginger (Gin), Cloves (Clov) and Amygdaline (Amyg), is one of the numerous complementary and alternative approaches as an anti-cancer therapy and holds great promise for cancer chemo-prevention with fewer side effects. The current study was designated to assess anti-tumoral immunity and anti-cancer and chemo-preventive effectiveness of herbal extracts of Cur, Ginger, Clov and Amyg in Ehrlich Ascites Carcinoma (EAC)-challenging mice. Chemo-preventive efficacy of herbal extracts of Cur, Gin, Clov and Amyg were analyzed in vivo by examination of the apoptosis rate of EAC tumor cells by flow cytometry. The total numbers of EAC cells, splenocytes counts and leucocytes count with their differentials relative % in peripheral blood (PB) of EACchallenging mice were investigated. EAC-challenging mice treated with herbal extracts of Cur, Gin, Clov and Amyg showed a marked decline in EAC tumor cell count and a noticeable increase in apoptosis rate of EAC tumor cells, a remarkable decrease in serum level of cancer antigen 125 (CA-125) with an obvious increase in the number of splenocytes comparing to that in EAC-challenging mice treated with PBS alone. Moreover, the data indicated an insignificant change in the total leucocytes count and their differentials relative % of eosinophil, neutrophils, monocytes and lymphocytes in EAC-challenging mice treated with Cur and Amyg, but these parameters were markedly increased in EAC-challenging mice injected with Gin and Clov compared to that in EAC-challenging mice treated with PBS alone. To conclude, the herbal extracts of Cur, Gin, Clov and Amyg may have anti-tumoral immunity and anti-cancer potency and potential to reduce the resistance to cancer conventional chemotherapy and exert cancer chemo-protective approaches with low adverse effects. Further research is necessary to determine the regimen's toxicity on various tissues and organs and to connect the diagnostic and therapeutic approaches used in the regimen's biomedical use.

Robust pancreatic tumor suppression by a novel combination treatment with anti-PD-1 immune checkpoint antibody and stroma modifying RNA oligonucleotide STNM01 in mice.

e14630 Background: Tumor stromal remodeling is an obstacle for immune checkpoint blockade therapies. STNM01 is a synthetic RNA oligonucleotide that represses carbohydrate sulfotransferase 15 (CHST15), which is responsible for tumor stromal remodeling. We have previously reported that STNM01 monotherapy by intratumoral injection repressed tumor stromal remodeling while increase tumor-infiltrating T lymphocytes (TILs) in pancreatic tumor-bearing mice. STNM01 was shown to increase TILs in patients with unresectable pancreatic cancer in a Phase I/IIa trial. In the present study, we investigated whether STNM01-mediated TIL enhancement improves anti-tumor effect of anti-PD-1 immune checkpoint antibody by combination treatment in murine models of pancreatic tumor. Methods: In mouse pancreatic cancer KPC and Pan02 subcutaneous syngeneic tumor models, mice were divided into 4 groups for treatment; 1) control, 2) STNM01 monotherapy, 3) anti-PD-1 antibody monotherapy, and 4) combination therapy with STNM01 and anti-PD-1 antibody. Intratumoral injections with 0.9-1.0 mg/mL of STNM01 or control and intraperitoneal administrations of anti-PD-1 antibody or control (5mg/kg) were then performed twice a week for 2 weeks. Mice were sacrificed after 2 week-treatment from baseline, and anti-tumor effects were evaluated by immunohistochemistry for KPC and flowcytometry for Pan02 model, respectively. Results: In the KPC-implanted syngeneic mouse model, combination treatment with intratumoral STNM01 and systemic anti-PD-1 antibody synergistically and robustly suppressed tumor growth as mean % tumor growth inhibition was over 80%. Tumor-infiltrating CD4+ and CD8+ T cells significantly increased in the anti-PD-1 plus STNM01 combination therapy group compared to anti-PD-1 monotherapy group. In the Pan02-implanted syngeneic mouse model, the combination treatment with STNM01 and anti-PD-1 significantly augmented necrosis area of tumor. Flowcytometry analyses showed that tumor-infiltrating CD3+, CD4+ and CD8+ T cells significantly increased in the anti-PD-1 plus STNM01 combination therapy group compared to control group. Although FoxP3+ Treg did not change, the ratio of CD8 to FoxP3 was significantly high only in the anti-PD-1 plus STNM01 combination therapy group. Notably, Ly6C+Ly6G+ granulocytic MDSCs dramatically diminished in the anti-PD-1 plus STNM01 combination therapy group, while anti-PD-1 monotherapy had no impact on tumoral MDSCs. Conclusions: The present work represents the first report of robust synergy between systemic anti-PD-1 antibody and single stroma modifying agent through intratumoral route of administration. Combination usage of STNM01 would provide a novel and clinically feasible therapeutic option to trigger remarkable effect of immune checkpoint inhibitors to this most hard-to-treat solid tumor.

Immunogenicity Analysis and Identification of Potential T-Cell Epitopes in C129R Protein of African Swine Fever Virus.

The highly conserved C129R protein of AFSV was utilized in the development of an ASFV recombinant adenovirus vaccine, demonstrating strong immunogenicity. In this study, we immunized 6-week-old female C57BL/6J mice via subcutaneous injection with 10 μg of purified C129R protein. Humoral and cellular immune effects were assessed using ELISA, flow cytometry, and ELISpot assays. Additionally, 19 peptides of the C129R protein were synthesized and screened for the use of bioinformatics. Positive T-cell epitopes were screened using ELISpot. The results indicated a higher proportion of CD4+ and CD8+ T lymphocytes in immunized mice compared to control mice. ELISA analysis revealed a serum titer of approximately 1:1, 638, 400 in the experimental group of mice. Additionally, peptides C11(53-61aa), C14(81-89aa), C16(97-105aa), and C18(116-124aa) from the C129R protein were able to activate mice spleen lymphocytes to produce IFN-γ. These findings suggest that the C129R protein significantly enhances both humoral and cellular immunity in immunized mice. Moreover, peptides C11, C14, C16, and C18 may serve as potential T-cell epitopes for the C129R protein. These results lay the groundwork for the further exploration of ASFV C129R protein and the identification of novel ASF vaccine antigens.

Metformin enhances the anti-tumor effects mediated by attenuated Salmonella typhimurium

In bacterial-based cancer therapy, while attenuated Salmonella treatment effectively inhibits tumor progression, single bacterial therapy struggles to completely suppress tumor growth and metastasis. Metformin, a natural compound derived from Galega officinalis L. and known for its antidiabetic properties, also demonstrates significant anti-tumor activity. However, the combined effect of metformin and attenuated Salmonella on tumor treatment has not been thoroughly investigated. In this study, we observed that metformin enhances attenuated Salmonella-mediated recruitment of tumor-infiltrating immune cells, such as NK cells, neutrophils, M1 macrophages, CD4+, and CD8+T lymphocytes in CT26 tumor-bearing mice. Furthermore, the combined treatment of metformin and attenuated Salmonella increases the protein expression of PEN2, PD1, and PD-L1. This finding is likely associated with the enhanced anti-tumor effects of S.t ΔppGpp by metformin, indicating that the combination of S.t ΔppGpp and metformin holds promise as a synergistic strategy for attenuated Salmonella-based immunotherapy.

CD3 aptamers promote expansion and persistence of tumor-reactive T cells for adoptive T cell therapy in cancer

The CD3/TCR complex is responsible for antigen-specific pathogen recognition by T cells, and initiates the signaling cascade necessary for activation of effector functions. CD3 agonistic antibodies are commonly used to expand T lymphocytes in a wide range of clinical applications, including in adoptive T-cell therapy for cancer patients. A major drawback of expanding T-cell populations ex vivo using CD3 agonistic antibodies is that they expand and activate T cells independent of their TCR antigen specificity. Therapeutic agents that facilitate expansion of T cells in an antigen-specific manner and reduce their threshold of T-cell activation, are therefore of great interest for adoptive T-cell therapy protocols. To identify CD3-specific T-cell agonists, several RNA aptamers were selected against CD3 using SELEX combined with high-throughput sequencing. The extent and specificity of aptamer binding to target CD3 were assessed through SPR, P32 double filter assays and flow cytometry. Aptamer-mediated modulation of the threshold of T-cell activation was observed in vitro and in pre-clinical transgenic TCR mouse models. The aptamers improved efficacy and persistence of adoptive T-cell therapy by low affinity TCR-reactive T lymphocytes in melanoma-bearing mice. Thus, CD3-specific aptamers can be applied as therapeutic agents which facilitate the expansion of tumor-reactive T lymphocytes while conserving their tumor specificity. Furthermore, selected CD3 aptamers also exhibit cross-reactivity to human CD3, expanding their potential for clinical translation and application in the future.

Effects of Partial-Body, Continuous/Pulse Irradiation at Dose Rates from FLASH to Conventional Rates on the Level of Surviving Blood Lymphocytes: Modeling Approach II. Two- and Multiple-Pulse Irradiation.

Mathematical models, which describe effects of partial-body, two- and multiple-pulse irradiation at high total doses D and at average dose rates N from FLASH to conventional rates on the level of surviving blood lymphocytes in humans and mice, have been developed originating in the previously proposed approach. These models predict that levels of surviving blood lymphocytes in humans and mice increase with increasing the dose rate from N=D/TR (TR is the time of the blood flowing into or out of the irradiated segment of the blood circulatory system) to FLASH rates and approach an upper limiting level equal to (1-vR), where vR is the fraction of blood volume in the irradiated segment of the blood circulatory system. Levels of surviving blood lymphocytes computed at total doses D of 10-40 Gy and at average of dose rates N, which are equal to or exceed 40 Gy/s for humans and 400 Gy/s for mice, are nearly indistinguishable from the upper limiting level. These results can be interpreted as the models reproducing the optimal blood lymphocyte sparing in these mammals after such exposures. With decreasing the dose rate from N=D/TR to conventional rates, at multiple-pulse irradiation the levels of surviving blood lymphocytes in humans and mice decrease to lower limiting levels, whereas at two-pulse irradiation they change cyclically and do not fall below their values for the delivery time equal to TR. Additionally, effects of two- and multiple-pulse irradiation of the whole abdomen in mice on the level of surviving blood lymphocytes are simulated within the developed models. Regimens of two- and multiple-pulse irradiation are taken the same as those reported in experiments, where effects of such exposures on the level of surviving crypts in mice were studied. Juxtaposing the modeling results with the experimental data reveals that the level of surviving blood lymphocytes in mice after two- and multiple-pulse irradiation of the abdomen at average dose rates N from FLASH to conventional rates modulates the level of surviving crypts in these animals after such exposures. A hypothesis is proposed to explain this phenomenon.

Effects of Partial-Body, Continuous/Pulse Irradiation at Dose Rates from FLASH to Conventional Rates on the Level of Surviving Blood Lymphocytes: Modeling Approach. I. Continuous Irradiation.

A mathematical model developed by Cucinotta and Smirnova is extended to describe effects of continuous, partial-body irradiation at high doses D and at dose rates N from FLASH to conventional rates on the level of surviving blood lymphocytes in humans and small laboratory animals (mice). Specifically, whereas the applicability of the model is limited to the exposure times shorter than a single cardiac cycle T0, the extended model is capable of describing such effects for the aforementioned and longer exposure times. The extended model is implemented as the algebraic equations. It predicts that the level of surviving blood lymphocytes in humans and mice increases with increasing the dose rate from N= D/T0 to FLASH rates and approaches the upper limiting level of 1-vR, where vR is the fraction of blood volume in the irradiated part of the blood circulatory system. Levels of surviving blood lymphocytes computed at doses from 10 Gy to 40 Gy and at dose rates N, which equal or exceed 40 Gy/s for humans and 400 Gy/s for mice, are nearly indistinguishable from the upper limiting level. In turn, the level of surviving blood lymphocytes in humans and mice decreases with decreasing the dose rate from N= D/T0 to conventional rates and approaches a lower limiting level. This level strongly depends on the dose D (it is smaller at larger values of D) with a slight dependence on the dose rate N. The model with the parameters specified for mice (together with the model of the dynamics of lymphopoietic system in mice after partial-body irradiation) reproduce, on a quantitative level, the experimental data, according to which the concentration of blood lymphocytes measured in mice in one day after continuous, partial-body irradiation at a high dose and conventional dose rate is smaller at the larger value of vR. Additionally, the model predicts at the same high dose (>10 Gy) a faster restoration of the blood lymphocyte population in humans exposed to continuous, partial-body irradiation at a FLASH dose rate compared to a conventional dose rate.

Psychoneuroimmunomodulating effect of lymphocytes with ortho-fluoro-benzonal modulated activity in syngeneic long-term alcoholized recipients

IntroductionLymphocytes are dysfunctional during long-term ethanol consumption and may contribute the progression from healthy to problem drinking. GABAA receptors are molecular targets of ethanol on lymphocytes, potentiating the effects of alcohol.ObjectivesWe first demonstrated that original compound ortho-fluoro-benzonal, artificial GABA receptor ligand, has immunostimulating properties and is able to restored long-term alcoholized mice lymphocytes activity in vitro through GABAA receptors. Based on the previous results we investigated effects of the ex vivo ortho-fluoro-benzonal modulated lymphocytes in recipients with experimental alcoholism.MethodsMale (CBAxC57Bl/6)F1 mice with 6-month 10% ethanol exposure were undergoing the transplantation of syngeneic long-term alcoholized mice lymphocytes, pretreated in vitro with ortho-fluoro-benzonal. Recipient’s ethanol consumption, parameters of the nervous and immune systems functional activities were estimated.ResultsIt was shown that lymphocytes modulated ex vivo with ortho-fluoro-benzonal after intravenous injection caused in syngeneic long-term alcoholized recipients ethanol consumption decrease and stimulation of behavioral activity in the “open field” test against the background of changes in the level of a number of cytokines in pathogenetically significant brain structures. Stimulation of humoral immune response, estimated by the relative number of antibody-forming spleen cells was also detected in recipients after lymphocytes transplantation. The injected immune cells were recorded in the parenchyma of the spleen and brain of recipients, which suggests, in particular, their direct influence on these functions.ConclusionsResults demonstrated that transplantation of ortho-fluoro-benzonal-modulated lymphocytes caused positive psychoneuroimmunomodulating effect in long-term alcoholized recipients, which makes it possible to consider adoptive immunotherapy as a promising method in the treatment of alcoholism.Disclosure of InterestNone Declared

Recombinant 60-kDa heat shock protein from Paracoccidioides brasiliensis induces the death of mouse lymphocytes in a mechanism dependent on Toll-like receptor 4 and tumor necrosis factor.

Paracoccidioides fungi are thermodimorphic microorganisms that cause paracoccidioidomycosis (PCM), an autochthonous disease from Latin America, with most cases in Brazil. Humans become infected by inhaling conidia or mycelial fragments that transform into yeast at body temperature. These fungi cause chronic-granulomatous inflammation, which may promote fibrosis and parenchyma destruction in the lungs. In response to stress imposed by the host, fungi Paracoccidioides spp. increase the expression of heat shock proteins (HSP), which protect them by sustaining cellular proteostasis. Our group has studied the role of HSP60 in PCM, and previous data show that the recombinant HSP60 (rHSP60) has a deleterious effect when used in a single dose as therapy for experimental PCM. Here, we investigated the mechanism by which rHSP60 could worsen the disease. We found that rHSP60 caused the viability loss of splenic or lymph node cells from both immunized and non-immunized mice, including in splenic T lymphocytes under polyclonal stimulation with concanavalin A, probably by undergoing apoptosis. Among analyzed splenic cells, lymphocytes were indeed the main cells to die. When we investigated the death mechanisms, remarkably, we found that there was no viability loss in rHSP60-stimulated splenic cells from mice deficient in Toll-like receptor 4, TRIF adapter protein, and TNF receptor 1(TNFR1), as well as rHSP60-stimulated WT cells incubated with anti-TNF antibody. Besides, caspase-8 inhibitor IETD-CHO blocked the rHSP60 effect on splenic cells, suggesting that rHSP60 induces the extrinsic apoptosis pathway dependent on signaling via TLR4/TRIF and TNFR1.

Specific deletion of Mettl3 in IECs triggers the development of spontaneous colitis and dysbiosis of T lymphocytes in mice.

The enzymatic core component of m6A writer complex, Mettl3, plays a crucial role in facilitating the development and progress of gastric and colorectal cancer (CRC). However, its underlying mechanism in regulating intestinal inflammation remains unclear and poorly investigated. First, the characteristics of Mettl3 expression in inflammatory bowel diseases (IBD) patients were examined. Afterward, we generated the mice line with intestinal epithelial cells (IECs)-specific deletion of Mettl3 verified by various experiments. We continuously recorded and compared the physiological status including survival rate etc. between the two groups. Subsequently, we took advantage of staining assays to analyze mucosal damage and immune infiltration of Mettl3WT and Mettl3KO primary IECs. Bulk RNA sequencing was used to pursuit the differential expression of genes (DEGs) and associated signaling pathways after losing Mettl3. Pyroptosis-related proteins were to determine whether cell death was caused by pyroptosis. Eventually, CyTOF was performed to probe the difference of CD45+ cells, especially CD3e+ T-cell clusters after losing Mettl3. In IBD patients, Mettl3 was highly expressed in the inner-nucleus of IECs while significantly decreased upon acute intestinal inflammation. IECs-specific deletion of Mettl3 KO mice triggered a wasting phenotype and developed spontaneous colitis. The survival rate, body weight, and intestinal length observed from 2 to 8 weeks of Mettl3KO mice were significantly lower than Mettl3WT mice. The degree of mucosal damage and immune infiltration in Mettl3KO were even more serious than in their WT littermate. Bulk RNA sequencing demonstrated that DEGs were dramatically enriched in NOD-signaling pathways due to the loss of Mettl3. The colonic epithelium was more prone to pyroptosis after losing Mettl3. Subsequently, CyTOF revealed that T cells have altered significantly in Mettl3KO. Furthermore, there was abnormal proliferation of CD4+ T and markedly exhaustion of CD8 + T in Mettl3KO mice. In severe IBD patients, Mettl3 is located in the inner-nucleus of IECs and declined when intestinal inflammation occurs. Subsequently, Mettl3 prevented mice from developing colitis.

Effect of Solanum aculeastrum on hematological parameters of Al-bino mice infected with Aspergillus fumigatus

The goal of the current study was to research the changes in hematological parameters: WBC count, RBCs count, Hb, PCV, neutrophil, lymphocyte, and monocyte in albino mice infected with Aspergillus fumigatus by intraperitoneal injection after induced immunosuppression by intraperitoneal injection of cortisone. The current research also examined an attempt to reduce the infection load by treating Solanum aculeastrum. The result shows higher decreased significance (P≤0. 05) in RBCs, Hb, and PCV after being infected with A. fumigatus 7. 1 ± 0. 8, 11. 3 ± 0. 5 and 41. 5 ± 2. 4, respectively, while the total WBC count, neutrophil, lymphocyte, and monocytes were increased significantly (P≤0. 05) after treatment with S. aculeastrum in groups infected with A. fumigatus, compared to other groups. According to these results, we conclude that the alcoholic extract of S. Astrum has significant therapeutic and antifungal characteristics that lead to an increase in the total WBC count and, therefore, is considered a necessary alternative therapy for increasing immunity. Keywords: Cortisone, Hematology, Fungi, Iraq.

Effect of Solanum aculeastrum on hematological parameters of Al-bino mice infected with Aspergillus fumigatus

The goal of the current study was to research the changes in hematological parameters: WBC count, RBCs count, Hb, PCV, neutrophil, lymphocyte, and monocyte in albino mice infected with Aspergillus fumigatus by intraperitoneal injection after induced immunosuppression by intraperitoneal injection of cortisone. The current research also examined an attempt to reduce the infection load by treating with Solanum aculeastrum. The result shows higher decreased significance (P≤0. 05) in RBCs, Hb, and PCV after being infected with A. fumigatus 7. 1 ± 0. 8, 11. 3 ± 0. 5 and 41. 5 ± 2. 4, respectively, while the total WBC count, neutrophil, lymphocyte, and monocytes were increased significantly (P≤0. 05) after treatment with S. aculeastrum in groups infected with A. fumigatus, compared to other groups. According to these results, we conclude that the alcoholic extract of S. Astrum has significant therapeutic and antifungal characteristics that lead to an increase in the total WBC count and, therefore, is considered a necessary alternative therapy for increasing immunity. Keywords: Cortisone, Hematology, Fungi, Iraq.

Pseudorabies Virus HLJ Strain Suppresses Peripheral Blood T and B Lymphocytes in Mice

Background: Pseudorabies virus (PRV), responsible for pseudorabies, is one of the most important pathogen in swine industry. PRV infection suppresses interferon-β (IFN-β) and tumor necrosis factor-α (TNF-α) expression, which consequently results in an immune escape in the host. Methods: In this study, distribution of peripheral blood T and B cell sub-populations was analyzed by flow cytometry after infecting mice with 1×105 TCID50 PRV. Result: We found that the levels of CD3+ T, CD4+ T and CD8+ T lymphocyte in infection group were significantly decreased at 24 hpi (p less than 0.01) and remained at a low level until 72 hpi compared to the control group. Interestingly, the levels of CD19+ T lymphocyte were significantly lower after 24 hpi (p less than 0.01) and lasted until 48 hpi, then increased sharply and then dropped at 96 hpi. Collectively, these results indicated that PRV infection could play a negative role in adaptive immunity in mice by decreasing the levels of B and T lymphocytes.