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Summary Fever is a complex physiological response enhancing immune surveillance during infection and inflammation. Fever-range whole-body hyperthermia (WBH) treatment can experimentally mimic the febrile condition in mice. Here, we describe a protocol for the treatment of mice with WBH and normothermia. We describe the isolation of T cells from mouse spleen followed by the evaluation of T-cell adhesion and transmigration. This animal model can be applied to studying the dysfunction of the immune system induced by fever. For complete details on the use and execution of this protocol, please refer to Lin et al. (2019)., Graphical abstract, Highlights • Whole-body hyperthermia (WBH) can mimic the febrile condition in mice • We isolate T cells from WBH- or normothermia-treated mice • T-cell adhesion and transmigration assays show dysfunctions caused by fever, Fever is a complex physiological response enhancing immune surveillance during infection and inflammation. Fever-range whole-body hyperthermia (WBH) treatment can experimentally mimic the febrile condition in mice. Here, we describe a protocol for the treatment of mice with WBH and normothermia. We describe the isolation of T cells from mouse spleen followed by the evaluation of T-cell adhesion and transmigration. This animal model can be applied to studying the dysfunction of the immune system induced by fever.

Spleen is one of the important organs for studying graft-versus-host disease development using cryo-imaging modality. Up to this point, there is no algorithm developed to segment spleen tissues in the cryo-imaging data. In this study, we developed a new, automatic segmentation algorithm for spleen tissues in the cryo-images for the first time. The algorithm consisted of (1) pre-processing, (2) predicting, and (3) post-processing. The predicting model consisted of two U-Nets to separate spleen from background and white pulp from red pulp. The results generated by the algorithm were compared against the ground truth generated by cryo-imaging experts. The Dice similarity coefficients were about 86% for red pulp class and 87% for white pulp class. We also used the algorithm to measure spleen volumes and performed the T-cells proliferation assays on the results. We found that there was a significant difference between ratios of white pulp volume to spleen volume between allogeneic spleen and syngeneic spleen. The results were consistent with the previous biological reports. In conclusion, the algorithm accurately and objectively segmented the spleen tissues with similar quality to the experts. By incorporating the algorithm into the T-cell proliferation assay workflow, it should greatly increase throughput of the process as compared to manual segmentation by humans. We expect that this algorithm will help accelerate the research for further understanding of graft-versus-host disease and development of the cure.

We successfully developed a liver-like organ from a stroma-engineered mouse spleen with implanted human liver cells., Regenerating human organs remains an unmet medical challenge. Suitable transplants are scarce, while engineered tissues have a long way to go toward clinical use. Here, we demonstrate a different strategy that successfully transformed an existing, functionally dispensable organ to regenerate another functionally vital one in the body. Specifically, we injected a tumor extract into the mouse spleen to remodel its tissue structure into an immunosuppressive and proregenerative microenvironment. We implanted autologous, allogeneic, or xenogeneic liver cells (either primary or immortalized), which survived and proliferated in the remodeled spleen, without exerting adverse responses. Notably, the allografted primary liver cells exerted typical hepatic functions to rescue the host mice from severe liver damages including 90% hepatectomy. Our approach shows its competence in overcoming the key challenges in tissue regeneration, including insufficient transplants, immune rejection, and poor vascularization. It may be ready for translation into new therapies to regenerate large, complex human tissue/organs.

A novel polysaccharide FVPB2 was purified from fruiting bodies of Flammulina velutipes. Its structure was elucidated by monosaccharide composition and methylation analyses, UV-Visible and FTIR spectroscopy as well as NMR. FVPB2 was a homogeneous heteropolysaccharide (molecular weight ~ 1.50 × 104 Da) containing D-galactose, D-mannose, L-fucose, and D-glucose at molar ratio of 1.9:1.2:1:2.5. In vitro immunomodulatory studies showed FVPB2 induced proliferation of mouse spleen lymphocytes in a dose-dependent manner. The levels of IgM and IgG, secreted by B cells, increased after FVPB2 treatment. So FVPB2 has potential to be a new important immunomodulatory nutraceutical.

Mass cytometry, also called cytometry by time-of-flight (CyTOF), is an emerging powerful proteomic analysis technique that utilizes metal chelated polymer (MCP) as mass tags for interrogating high-dimensional biomarkers simultaneously on millions of individual cells. However, under the typical polymer-based mass tag system, the sensitivity and multiplexing detection ability has been highly restricted. Herein, a new structure mass tag based on a nanometal organic framework (NMOF) is reported for multiparameter and sensitive single-cell biomarker interrogating in CyTOF. A uniform-sized Zr-NMOF (33 nm) carrying 105 metal ions is synthesized under modulator/reaction time coregulation, which is monodispersed and colloidally stable in water for over one-year storage. On functionalization with an antibody, the Zr mass tag exhibits specific molecular recognition properties and minimal cross-reaction toward nontargeted cells. In addition, the Zr-mass tag is compatible with MCP mass tags in a multiparameter assay for mouse spleen cells staining, which exploits four additional channels, m/z = 90, 91, 92, 94, for single-cell immunoassays in CyTOF. Compared to the MCP mass tag, the Zr-mass tag provides an additional fivefold signal amplification. This work provides the fundamental technical capability for exploiting NMOF-based mass tags for CyTOF application, which opens up possibility of high-dimensional single-cell immune profiling, low abundant antigen detection, and development of new barcoding systems.

This study was conducted to assess the in vivo and ex vivo immunomodulatory effect of the ethanol leaves extract of Moringa peregrina in Balb/c mice. For this study, five groups of 5 Balb/c mice were given a single acute subtoxic oral dose of the ethanolic extract at 1.13, 11.30, 23.40 and 113.4 mg/kg and the immunomodulatory effect was assessed on the 6th day following the ingestion. In the (non-functional) assessment, the effect of the extract on the body weight, relative lymphoid organ weight, splenic cellularity and peripheral blood hematologic parameters were evaluated. While in the immunomodulation assessment (functional), we investigated the effect of the extract on the proliferative capacity of splenic lymphocytes and peripheral T and B lymphocytes using mitogen blastogenesis, mixed allogeneic MLR and IgM-Plaque forming cells assays. The ingestion of M. peregrina extract caused a significant increase in the body weight, weight and number of cells of spleen and lymph nodes of the treated mice. Furthermore, the count of RBCs, WBCs, platelets, hemoglobin concentration and PCV % were increased by the extract treatment in a dose-dependent manner. M. peregrina enhanced the proliferative responses of splenic lymphocytes for both T cell and B-cell mitogens. Likewise, the mixed lymphocyte reaction MLR assay has revealed a T-cell dependent proliferation enhancement in the extract treated mice. Moreover, the oral administration of M. peregrina leaves extracts significantly increased PFCs/10 6 splenocytes in a dose-dependent manner. In conclusion, subtoxic acute doses of M. peregrina extract demonstrated significant potential as an immunomodulatory agent even at the lowest dose of 1.13 mg/kg.

CD49b is a member of the integrin family, expressed on basophils, natural killer (NK) cells and a subset CD4(+) T cells in the spleen. This protocol describes the adoptive transfer of basophil-enriched CD49b(+) cells obtained from mouse spleens by magnetic enrichment. This protocol can be used to assess the contribution of basophils or basophil-derived mediators to a certain immune response.

Background and aim Sodium nitrite (NaNO2) is an inorganic salt with numerous applications in a variety of industries, as well as in medicine. Nevertheless, exposure to high levels of NaNO2 is toxic for animals and humans. Sodium nitrite intoxication is shown to decrease the activity of major antioxidant defence enzymes which is dependent on the maintenance of specific ion equilibrium. The aim of the present study was to investigate the effect of acute NaNO2 intoxication on the content of the essential metals iron (Fe), calcium (Ca) and zinc (Zn) in mouse spleen. Methods Mature male ICR mice were divided into four groups and subjected to acute NaNO2 exposure by a single intraperitoneal injection of 120 mg/kg body weight. Animals in each group were sacrificed at certain time interval after treatment (1 h, 5 h, 1 day and 2 days). Spleens were excised and processed for atomic absorption spectrometry analysis of Fe, Ca and Zn content. Results At the first hour after treatment, a decrease in Fe and Ca levels was observed. One day following NaNO2 administration, Zn concentration reached its lowest value and Ca levels remained lower, compared to the untreated controls. In contrast, Fe concentration increased on the first and second day after treatment. Conclusion The results of the present study demonstrate that acute NaNO2 intoxication provokes changes in the endogenous levels of Fe, Ca and Zn in mouse spleen. These findings suggest disruption of the ionic balance and impact on the activity of antioxidant defence enzymes.

Natural killer (NK) cells are members of a rapidly expanding family of innate lymphoid cells (ILCs). While most previously studied NK cells were derived from the mouse spleen and circulate in the blood, recently others and we found tissue-resident NK (trNK) cells in many tissues that resemble group 1 ILCs (ILC1s). During pregnancy, NK cells are the most abundant lymphocytes in the uterus at the maternal-fetal interface and are involved in placental vascular remodeling. Prior studies suggested that these uterine NK (uNK) cells are mostly derived from circulating NK cells. However, the murine virgin uterus contains mostly trNK cells and it has been challenging to determine their contribution to uNK cells in pregnancy as well as other potential function(s) of uNK cells due to the dynamic microenvironment in the pregnant uterus. This review focuses on the origins and functions of the heterogeneous populations of uNK cells during the course of murine pregnancy.

Acanthopanax senticosus is an herb that has been used as a traditional remedy and medicine source. Its anti-inflammatory and, anti-oxidative effects have been reported in previous studies. This study aimed to investigate the effect of Acanthopanax senticosus water extracts on mouse macrophage cell in vitro. Mouse splenocyte proliferation increased after application of Acanthopanax senticosus water extract supplement of 5, 10, 50, 100, 250, 500, 1,000 μg/mL after 48 h pre-treatment with a mitogen (ConA or LPS). The production of cytokines secreted by LPS and non LPS stimulated macrophages was detected by ELISA assay using a cytokine kit. Cytokine production (IL-2, IFN-γ, and TNF-α) increased after water extract supplementation. The result of this in vitro study, showed that splenocyte proliferation and cytokine production by activated peritoneal macrophages were increased after Acanthopanax senticosus water extract in the range of 500~1,000 μL/mL. Thus, it is suggested that supplementation with Acanthopanax senticosus water extracts may enhance immune function by regulating splenocyte proliferation and enhancing cytokine production by activated macrophage.Key words: splenocyte proliferation, Acanthopanax senticosus, macrophage, cytokine, immune

Background and aims Skin injury is the second most common clinical manifestation in patients with systemic lupus erythematosus (SLE). Oestrogen may affect the onset and development of SLE. This study was undertaken to elucidate the role of oestrogen in the development of SLE skin injury. Methods We investigated the role of oestrogen and its membrane receptor GPER1 in SLE-related skin injury in mice treated with SLE serum in vivo, and monocytes from mouse spleen in vitro. Results We found that skin injury induced by SLE serum was more severe in female mice and required monocytes. E2 promoted these effects through the membrane receptor GPER1 located in lipid rafts and that inhibition of lipid rafts and GPER1 suppressed SLE serum-induced skin inflammation and expression of inflammatory molecules. Conclusions We conclude that oestrogen promotes the development of skin injury induced by SLE serum through the membrane receptor GPER1 and that lipid rafts play an important role in the regulatory effect of GPER1 in SLE skin inflammation.

It has been reported that water at the interface of a hydrophilic thin film forms an exclusion zone, which has a higher density than ordinary water. A similar phenomenon was observed for a hydrated hydrophilic ceramic powder, and water turns into a three-dimensional cell-like structure composed of high density water and low density water. This structured water appears to have a stimulative effect on plant growth. This report outlines our study of antioxidant properties of this structured water and its effect on cell bioactivities. Culturing media which were prepared utilizing this antioxidant structured water promoted the viability of RAW 264.7 macrophage cells by up to three times. The same tendency was observed for other cells including IEC-6, C2C12, and 3T3-L1. Also, the cytokine expression of the splenocytes taken from a mouse spleen increased in the same manner. The water also appears to suppress the viability of cancer cell, MCF-7. These results strongly suggest that the structured water helps the activities of normal cells while suppressing those of malignant cells.

In the experiment, a systematic study on the genotoxicity of organic extracts of the water in a reservoir at wet season and dry season. And the study was done by using comet assay. The study was to appraise the waters qualities after every treatment step from the view of the DNA damages on the mouse spleen lymphocytes. The results of the experiment can provide reliable basis for adjusting the treatment reasonably to purify raw water of different qualities.

We observed degradation of β-actin mRNA and 18S rRNA in mouse spleen cells under constant temperature conditions in the different temperature group during postmortem intervals (PMIs) of 0–72 h. Thirty-nine mice were sacrificed by cervical dislocation and kept at constant temperatures of 10°C, 15°C, 20°C, 25°C, and 30°C. From 0 to 72 h after death, total RNA in spleen cells was extracted every 6 h. The cycle threshold (Ct) values of β-actin mRNA and 18S rRNA were obtained by real-time-quantitative polymerase chain reaction. The results showed that, under the conditions of different and constant temperatures after mouse death at 72 h, the Ct values of β-actin and 18S, Ct ratios of β-actin to 18S, and relative ratios of β-actin to 18S were significantly correlated with PMI. In addition, the relative degradation rates of β-actin and 18S appeared to change from fast to slow with the increase of temperature. By interpolation and fitting analysis of the data, we obtained a ternary quintic equation of the relationship between the change in the relative ratios and PMI, which can be used to infer PMI within a certain temperature range (10°C–30°C).

A part of his ‘theory of haematopoiesis’, Alexander Maksimov first proposed the existence of stem cells circa 1908. The importance and role of stem cells remained shrouded until the early 1960s when Becker and colleagues demonstrated self-renewing haematopoietic cells in mouse spleen [1], and Joseph Altman first examined neurogenesis in the mammalian adult brain [2]. In the 1980s, two groups simultaneously reported the ability to i i h t t t a w a a v d r o c d t l i i t s t m m c a u

The present study showed that a hot water extract of the fruiting body of the edible mushroom Boletopsis leucomelas, known as ‘Kurokawa’ Japanese, strongly stimulated IgA-production in mouse spleen cells in our screening experiment. The in vivo study was also conducted with the objective of enhancing adaptive immune response by oral administration of the hot water extract of B. leucomelas (BLE) in lipopolysaccharide (LPS)-challenged mice. The mice were fed a standard diet with or without 0.16% BLE. The mice were also orally administered sodium phosphate buffer or LPS weekly at days 7, 14 and 21. Results indicated that LPS-specific serum IgG, IgM and IgA were increased in the BLE diet group compared to the standard diet group. Interestingly, intestinal total IgA and LPS-specific IgA were significantly increased in the BLE diet group. Moreover, the

The distribution of triterpene glycoside cucumarioside A₂-2, the main compound of medical lead Cumaside in immunodeficiency diseases, in mouse spleen was determined. For this purpose the stability and dynamics of glycoside content changes over time in Balb/c mouse spleen tissue homogenate as well as the study of the cucumarioside A2-2 spatial distribution in tissue sections were investigated using radiospectroscopy, MALDI-MS and MALDI Imaging Mass Spectrometry (IMS), correspondingly. Cucumarioside A₂-2 is reliably detected by MALDI-MS in the mouse spleen tissue after single intraperitoneal (i.p.) injection at a dosage of 5 mg/kg. The glycoside is stable in the spleen and does not undergo metabolic transformation in either tissue homogenates or in the intact organ within 24 h after i.p. injection. The cucumarioside A₂-2 was absorbed fairly rapidly: the glycoside maximum concentration (Cmax) in tissue homogenate was observed in the first 30 min after injection; the minimum values were registered in 3 h. These results are in agreement with those obtained in the pharmacokinetic study of (3)H-cucumarioside A₂-2. It was established by MALDI-IMS that glycoside was mainly located in the tunica serosa part of the spleen and only a small amount was detected within the red and white pulp of the organ. MALDI MS images obtained 15-30 min post dosage clearly reflect high drug concentrations in the regions surrounding the organ followed by its decline in the surface part and a very slight redistribution to the internal part of the spleen.

생리활성물질을 다량함유하고 있는 마늘의 발효산물인 흑마늘의 면역활성을 검증하기 위하여 C57BL6 마우스 비장세포를 이용하여 흑마늘이 비장세포의 활성화에 미치는 영향을 확인하였다. 흑마늘 추출물은 시판되는 남해 흑마늘 액기스를 농축하여 사용하였다. 그 결과 IL-2에서 흑마늘 추출물만 처리한 군에서 생성이 증가하였으며, LPS와 흑마늘 추출물을 함께 처리하였을 때 IL-2와 TNF- ${\alpha}$ , IFN- ${\gamma}$ 의 생성이 LPS만 처리한 군보다 증가하여 대식세포나 T림프구의 발현에 의해 일어나는 세포성 매개 면역을 활성화를 유도하는 Th1 세포의 발현을 활성화 하였다. 그리고 IL-6는 흑마늘 추출물만 처리하였을 때 후기생성이 증가하였으며, LPS와 흑마늘 추출물을 함께 처리한 경우 LPS만 처리한 군보다 IL-4와 IL-6의 생성이 증가하였다. IL-10은 LPS와 흑마늘 추출물을 함께 처리하였을 때 후기 생성이 감소하였는데, 이는 B 림프구의 활성화에 따른 항체생성 면역을 활성화하며 Th1 세포로부터 유도되는 세포성 면역반응을 억제함으로서 항체유도 체액성 면역반응으로 전환을 효과적으로 조절하는 것을 확인하였다. 따라서 흑마늘 추출물은 마우스 비장세포에서 T 림프구의 활성화에 따른 Th1 세포와 Th2 세포가 활성화되어 면역계의 세포성 면역과 체액성 면역반응을 활성화하여 면역조절에 효과를 나타내는 것으로 사료된다. 【The effect of black garlic extract on the activation of spleen cells from a C57BL6 mouse was investigated to examine immune activities of of fermented black garlic containing a variety of bioactive substances. xtract obtained from the concentration of commercial Namhae black garlic was used for the analysis of immune activities. Treatment with the extract increased the expression of interleukin-2 (IL-2) cytokine. The simultaneous administration of the extract plus lipopolysaccharide (LPS) increased the expression of IL-2, tumor necrosis factor (TNF)- ${\alpha}$ , and interferon (IFN)- ${\gamma}$ compared with that of a control group. This result suggests that cellular immunity can be induced by macrophages, resulting in the expression of T lymphocytes and T helper type 1 (Th1) cells. In addition, treatment with the extract increased the late response of IL-6 cytokines, and the extract plus LPS augmented the expression of IL-4 and IL-6 compared with that of an LPS-treated group. Meanwhile, the extract plus LPS decreased the late response of IL-10, suggesting that humoral immunity can be activated by stimulating B lymphocytes, suppressing cellular immunity, and effectively modulating the conversion into humoral immune responses. These findings demonstrate that the black garlic extract activates Th1 and Th2 cells by stimulating T lymphocytes in mouse spleen cells and leads to immunomodulation by activating cellular and humoral immune responses of the immune system.】

Splenic transitional B-cells (T1 and T2) are selected to avoid self-reactivity and to safeguard against autoimmunity, then differentiate into mature follicular (FO-I and FO-II) and marginal zone (MZ) subsets. Transcriptomic analysis by RNA-seq of the five B-cell subsets revealed T1 cell signature genes included RAG suggesting a potential for receptor revision. T1 to T2 B-cell differentiation was marked by a switch from Myb to Myc, increased expression of the PI3K adapter DAP10 and MHC class II. FO-II may be an intermediate in FO-I differentiation and may also become MZ B-cells as suggested by principal component analysis (PCA). MZ B-cells possessed the most distinct transcriptome including downregulation of CD45 phosphatase-associated protein (CD45-AP/PTPRC-AP), as well as upregulation of IL-9R and innate molecules TLR3, TLR7 and bactericidal Perforin-2 (MPEG1). Among the endosomal TLRs, stimulation via TLR3 further enhanced Perforin-2 expression exclusively in MZ B-cells. Using gene-deleted and overexpressing transgenic mice we show that IL-9/IL-9R interaction resulted in rapid activation of STAT1, 3 and 5, primarily in MZ B-cells. Importantly, CD45-AP mutant mice had reduced transitional and increased mature MZ and FO B-cells, suggesting that it prevents premature entry of transitional B-cells to the mature B-cell pool or their survival and proliferation. Together, these findings suggest, developmental plasticity among splenic B-cell subsets, potential for receptor revision in peripheral tolerance whereas enhanced metabolism coincides with T2 to mature B-cell differentiation. Further, unique core transcriptional signatures in MZ B-cells may control their innate features.

Monoclonal antibodies offer high specificity and this makes it an important tool for molecular biology, biochemistry and medicine. Typically, monoclonal antibodies are generated by fusing mouse spleen cells that have been immunized with the desired antigen with myeloma cells to create immortalized hybridomas. Here, we describe the generation of monoclonal antibodies that are specific to Chikungunya virus using ClonaCell-HY system.

Dendritic cells (DCs) were discovered in 1973 by Ralph Steinman as a previously undefined cell type in the mouse spleen and are now recognized as a group of related cell populations that induce and regulate adaptive immune responses. Studies of the past decade show that, both in mice and humans, DCs are composed of subsets that differ in their localization, phenotype, and functions. These progresses in our understanding of DC biology provide a new framework for improving human health. In this review, we discuss human DC subsets in the context of their medical applications, with a particular focus on DC targeting.

To utilize ultraviolet (UV) irradiation as a means to prepare hypo-allergenic food, we investigated the molecular weight profile, secondary structure content, fluorescence spectrum, and immunoglobulin (Ig) production-regulating activity of β-lactoglobulin after UV-irradiation. UV-irradiation caused a change on the molecular size distribution, disruption of the ordered structure, and decrease of emission intensity of β-lactoglobulin. The alteration on Igs production regulating activity of β-lactoglobulin by UV-irradiation was observed in the mouse spleen lymphocytes system. These results suggest that UV-irradiation is effective for alteration of molecular properties and antigenicities of β-lactoglobulin and such treatment should be useful for the preparation of low allergenic foods.

Objective The aim of the study was to investigate the adaptive response of low-dose radiation-induced apoptosis in mouse spleen cells and its related proteins control mechanism.

Summary Through electron microscopic observations of the normal mouse spleen, the following findings were obtained concerning denucleation mechanism of the erythroblast: 1. In mice the nuclei of the erythroblasts are denucleated by phagocytic activity of the reticular cell, located in the center of the erythroblastic islets. In a physiologic state, this mechanism is considered to be playing the main role in denucleation. 2. The protoplasmic projection of the reticular cell compresses and makes an infolding between the head and body of the orthochromatic erythroblast, and finally the head is cut off and phagocytized by this reticular cell, leaving the body to develop into a reticulocyte.

Using comet assay, a statistically significant increase (p < 0.05) in the level of DNA breaks in spleen cells was revealed in male CBA/lac mice exposed to gamma-radiation (1.7 cGy/day) or 90Sr (150-250 Bq/day) for 210 days. The level of DNA breaks also increased under combined exposure to both gamma-radiation and 90Sr (p < 0.05), but to a lesser degree than under exposure to each of these factors alone. Upon additional in vitro treatment of spleen cells with hydrogen peroxide, the relative increase in the level of DNA breaks was smaller in cells of irradiated mice than in the control. The ratio of the level of DNA breaks after hydrogen peroxide treatment to that before this treatment in control mice was 4.2 +/- 0.9, compared to 1.4 +/- 0.6 in gamma-irradiated mice, 1.9 +/- 0.8 in 90Sr-irradiated mice, and 2.3 +/- 0.8 in mice exposed to both gamma- and 90Sr-irradiation.

Mouse spleen lymphocytes treated with rabbit IgG anti-sheep erythrocytes (SRBC) complexed with protein A of Staphylococcus aureus (SpA) form rosettes with SRBC. The attachment of SRBC to lymphocytes was due to the binding of the SpA-IgG antibody complex to the surface of the lymphocytes and was thus considered "arming" of the cells. Normal mouse spleen cells "armed" with SpA-rabbit IgG anti-chicken erythrocytes (CRBC) kill specifically 51Cr-labeled CRBC "in vitro" in the absence of free antibodies. The killing by these "armed" cells is an effect of the cell-bound SpA-IgG antibody complex. Both the SRBC rosette formation and the cell-mediated CRBC killing was dependent on the concentration of the SpA-IgG antibody complexes used for "arming" the cells. A 100-fold increase in rosette formation or in killing of target cells was recorded for lymphocytes treated with SpA-IgG antibody complexes in comparison with cells treated with noncomplexed IgG antibodies. The specific binding of SpA-IgG antibody complexes to the Fc receptors of mouse spleen cells was demonstrated by inhibition studies. More than 60% inhibition of the rosette formation and in the killing of target cells was shown for cells treated with normal rabbit IgG or its Fc fragment before addition of the SpA-IgG antibody complex.

In this study, we compared with 80% ethanol extracts from peel of Poncirus trifoliata (PTP) and peel of Citrus junos (CJP) against antioxidant and immune activities. Total phenolics and flavonoids contents in PTP extracts were 60.75±1.15 and 33.75±0.15 ㎎/100 g, respectively, and those were lower than CJP extracts. Antioxidant activities of PTP were increased with the more concentration, and were similar to CJP. Antioxidant activities of PTP were increased with increasing of concentration, and were similar to those of CJP. The NO production in macrophage cell lines were increased in a dose-dependent manner, until 5 ㎎/ml of CJP and 1 ㎎/ml of PTP compared with control cells, but decreased at higher concentrations. The proliferation of mouse spleen cells were increased in a dose-dependent manner, until 1 ㎎/ml of CJP and PTP compared with control cells but decreased at higher concentrations. The NO production in macrophage cell lines treated with PTP and CJP were increased in a dose-dependent manner, compared with untreated control cells until the concentrations of 1~5 ㎎/ml (CJP) and 1 ㎎/ml (PTP) but decreased at higher concentrations than that. The proliferation of mouse spleen cells treated with PTP and CJP were increased in a dose-dependent manner, compared with untreated control cells until the concentration of 1 mg/ml but decreased at higher concentrations than that.

To determine whether interferon gamma (IFN-gamma) can be used as a biomarker of exposure to viral pathogens, 12-week-old BALB/c mice were injected intraperitoneally with coxsackievirus B3 (CVB3) or coxsackievirus B4 (CVB4) diluted in sterilized phosphate-buffered saline (PBS). Control mice were injected with PBS only. Four months after viral infection, mouse spleen cells were harvested and assayed for the release of IFN-gamma by memory T cells after in vitro stimulation with viral antigens, phytohemagglutinin (PHA), and PBS, respectively. The level of IFN-gamma was examined by an antibody-capture enzyme-linked immunosorbent assay (ELISA). A marked increase in the level of IFN-gamma was observed when memory T cells from CVB3-infected mice were incubated with CVB3 virus, but not with CVB4 or PBS. Conversely, memory T cells from mice infected by CVB4 were not stimulated to produce IFN-gamma when they were incubated with CVB3 and PBS, but did significantly produce IFN-gamma when stimulated with CVB4. T cells from mice injected with PBS did not release IFN-gamma after stimulation with CVB3 or CVB4. However, these T cells did release IFN-gamma after stimulation with PHA. Our results demonstrated that IFN-gamma produced by memory T cells is virus-specific and may have use as a biomarker in viral exposure studies. The results of this study may be extended to the study of infection by pathogens that are capable of inducing cell-mediated immune response in humans.

Aim: To study the relationship between primary structures of oligodeoxynucleo-tides (ODN) containing unmethylated deoxycytidyl-deoxyguanosine (CpG) dinucleotide motifs and their immunostimulatory activities in mouse spleen cells. Methods: A series of CpG ODN with different primary structures were synthesized. Their capabilities to stimulate mouse spleen cell proliferation were determined by [3H]thymidine incorporation assay. Cytokine (interleukin [IL]-6, IL-12, and IFN-α) secretion spectra induced by CpG ODN were assessed by ELISA. The ability of CpG ODN to activate natural killer cells was evaluated by standard 4 h 51Cr-release assay. Flow cytometry was utilized to examine the expressions of various lymphocyte surface molecules on diverse immunocytes. An effective CpG ODN for murine, ODN1826, was set as the template of modification and the positive control. Results: The immunostimulatory activities of CpG ODN with different sequences and compositions varied markedly, both in character and in extent. It was useless for improving the immunostimulatory activity of ODN1826 by simply increasing the functional hexameric CpG motif number, modifying the site of CpG motifs, or changing the distance between multi-CpG motifs. However, an addition of a self-complementary palindrome structure at the 3′-end, but not the 5′-end of CpG ODN, aroused marked improvement in its activity. Several designed ODN had superior comprehensive immunostimulatory properties compared to ODN1826. Conclusion: The immunostimulatory activity of a CpG ODN was relevant to its primary structure. It was useless for promoting immunostimulatory activity to simply change CpG motif number, space, or distance. The 3′-end palindrome structure of CpG ODN is associated with enhanced immunostimulatory activity.

Quantitative features of the proteome are extremely useful for studying cellular processes at a molecular level. In this study, we attempted to construct quantitative reference proteome maps of the mouse spleen and lymph node based on 2-DE followed by protein identification using MS. We analyzed more than 1000 spots on the 2-DE images and consequently were able to determine that 919 spots were derived from 328 different genes. To obtain statistically reliable information of the protein levels from these 2-DE images, we measured the volumes of the respective spots on 2-DE images obtained by four to six independent experimental runs. These measurements were used to calculate the variability of the volumes of the respective spots on 2-DE following subcellular fractionation, which enabled us to discriminate differentially produced proteins from those within the range of intrinsic variability. More importantly, while the 2-DE data have been traditionally collected in a gel image-based manner, the resultant quantitative 2-DE data could be analyzed using the same procedure as that for mRNA expression profiles. This greatly assists in bridging the gap between the analyses of transcriptomes and proteomes and enables the integration of this data on the same informational platform.

A total number of 31 types of seaweed were assessed with regard to their effects on the proliferation of mouse spleen and thymus cells in a culture, using an MTT reduction assay. Acetone:dichloromethane (1∶1) extracts of three seaweed plants:Derbesia marina, Sargassum sp., andHisikia fuziformis, exhibited significantly positive effects on the survival of mouse spleen and thymus cellsin vitro. The acetone: dichloromethane (1∶1) extracts ofSargassum sp., in particular, much more potent effects on thymus cell activation than did any of the other types of seaweed. However, the methanol extracts ofSargassum ringgoldianium andChondrus crispus exerted a stimulatory influence only on the proliferation of mouse spleen cells, whereas the methanol extracts ofGrateloupia lanceolata exhibited significant cell proliferation properties in both spleen and thymus cells.

Theophylline (TH) is a methylxanthine widely used in clinical practice. The genotoxic effects of TH was investigated in mouse spleen, and spermatogonia cells. Also damage of DNA and molecular detection of apoptosis were applied. TH was administered orally as single dose of 25, 50, 75 and 120 mg kg−1 b.wt. and a multiple treatment with a daily dose of 25, 50 and 75 mg kg−1 b.wt. for 3 and 7 successive days. TH induced a significant increase in the percentage of chromosomal aberrations in somatic and germ cells which was dose and time dependent.Also, TH induced DNA damage and apoptosis in a dose and time dependent manner in mouse liver cells. However, induced DNA damage and apoptosis was maximally detected at a concentration of 120 mg TH kg−1 b.wt. after oral treatment for 1 d.

Group X secretory phospholipase A2 (sPLA2-X) and cytosolic phospholipase A2 alpha (cPLA2alpha) are involved in the release of arachidonic acid (AA) from membrane phospholipids linked to the eicosanoid production in various pathological states. Recent studies have indicated the presence of various types of cross-talk between sPLA2s and cPLA2alpha resulting in effective AA release. Here we examined the dependence of sPLA2-X-induced potent AA release on the cPLA2alpha activation by using specific cPLA2alpha or sPLA2 inhibitors as well as cPLA2alpha-deficient mice. We found that Pyrrophenone, a cPLA2alpha-specific inhibitor, did not suppress the sPLA2-X-induced potent AA release and prostaglandin E2 formation in mouse spleen cells. Furthermore, the amount of AA released by sPLA2-X from spleen cells was not significantly altered by cPLA2alpha deficiency. These results suggest that sPLA2-X induces potent AA release without activation of cPLA2a, which might be relevant to eicosanoid production in some pathological states where cPLA2a is not activated.

Antitumor activities of onion methanol extract (OME) and onion Kimchi methanol extract (OKME) were investigated by using aflatoxin -mediated Salmonella typhimurium mutagenicity and the model of cytotoxicity on the cancer cell lines. Their immune activities were also investigated by using mouse spleen cells and macrophage cell lines, respectively. OME and OKME showed the enhanced antimutagenicity in a dose-dependent manner in particular, the activity of OKME was higher than that of OME. OME and OKME decreased over 20% of the proliferation of the A549 (lung cancer cell) and MCF-7 (breast cancer cell) cell lines when compared with the control at 1,000 g/mL. The proliferation of mouse spleen cells and the NO production in marcrophage cell lines treated OME and OKME were increased in a dose-dependent manner compared with untreated control cells, and their activities of OKME were higher than those of OME.

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. However, its role in B cells remains unexplored. We here show a role for IL-27 in the induction of T-bet expression and regulation of Ig class switching in B cells. Expression of WSX-1, one subunit of IL-27R, was detected at the mRNA level in primary mouse spleen B cells, and stimulation of these B cells by IL-27 rapidly activated STAT1. IL-27 then induced T-bet expression and IgG2a, but not IgG1, class switching in B cells activated with anti-CD40 or LPS. In contrast, IL-27 inhibited IgG1 class switching induced by IL-4 in activated B cells. Similar induction of STAT1 activation, T-bet expression and IgG2a class switching was observed in IFN-γ-deficient B cells, but not in STAT1-deficient ones. The induction of IgG2a class switching was abolished in T-bet-deficient B cells activated with LPS. These results suggest that primary spleen B cells express functional IL-27R and that the stimulation of these B cells by IL-27 induces T-bet expression and IgG2a, but not IgG1, class switching in a STAT1-dependent but IFN-γ-independent manner. The IL-27-induced IgG2a class switching is highly dependent on T-bet in response to T-independent stimuli such as LPS. Thus, IL-27 may be a novel attractive candidate as a therapeutic agent against diseases such as allergic disorders by not only regulating Th1 differentiation but also directly acting on B cells and inducing IgG2a class switching.

We investigated the regulatory effects of noradrenalin (NA) and corticosterone (CS) on the antibody-forming response against ovalbumin (OVA) of cultured mouse spleen cells. SAMP1 and C3H/He mice were immunized with two intraperitoneal injections of OVA with alum adjuvant. Spleen cells primed to OVA were then cultured with OVA in the presence of varying concentrations of NA. NA showed significant dose-dependent regulatory effects on the response of the C3H/He spleen cells, augmenting and suppressing the anti-OVA antibody production of both IgGs and IgAs at concentrations of 3.0×10−5 and 3.0×10−4 M, respectively. In contrast, NA at any concentration tested showed little effect on the antibody response of SAMP1 spleen cells. CS suppressed the antibody response of C3H/He spleen cells in a dose-dependent manner between 1.0×10−7 and 1.0×10−5 M, but the antibody response of SAMP1 spleen cells was not significantly altered by the same concentration of CS. These results suggest that the T-helper-dependent antibody-forming system in SAMP1 mice has defective response to regulatory signals from the neuroendocrine system.

The effect of radiation on TK is more complicated than would be expected from earlier results on bone marrow cells (Feinendegen et al. 1984, Int. J. Radiat. Biol. 45, 205). TK activity increased at 0.01 Gy and then decreased up to 1 Gy in mouse spleen. In contrast to the results for the spleen, an increase in activity at 0.1 Gy was seen in mouse thymus. The activity of dephosphorylated TK1 (TK1a) in both spleen and thymus was reduced to 50% after irradiation at 0.5–1 Gy. The degree of phosphorylation (TK1b/TK1a ratio) changed in spleen, but not in thymus. The activity of TK2 in mouse liver increased at 3 h after 5 Gy by about 60%. In mouse ascites tumour, a dose-independent (1–5 Gy) oscillating TK1 activity was found up to 24 h, especially for TK1a and TK1b. The amount of TK1 was unchanged up to 12 h, but decreased at 24 h. This suggests that the differences in the changes in the degree of phosphorylation of TK1 after irradiation among spleen, thymus and ascites tumour further underline the complexity of the response of TK1 activity to irradiation. The dramatic change in the activities of TK1a and TK1b may illustrate that both of them are more radiosensitive than TK-h, a variant with mixed TK1 and TK2 properties.

We first examined two lines of transgenic mouse, gpt delta and Muta, for sensitivity of radiation-induced mutations in spleen. The gpt delta mouse could detect mutations induced by 2 to 8 Gy of gam...

AIMS This study aimed to identify the main active component of Lactobacillus brevis KB290 (KB290) that is responsible for enhanced cell-mediated cytotoxic activity of mouse splenocytes Live KB290, a probiotic strain derived from a Japanese traditional pickle, was previously reported to modulate innate immune responses as affecting on cell-mediated cytotoxic activity of mouse splenocytes. METHODS AND RESULTS We used live KB290, heat-killed KB290, a derivative strain (Lact. brevis KB392) with different amounts of cell-bound exopolysaccharide (EPS-b), and a crude extract of EPS-b from KB290 cell surface. Female BALB/c mice were fed a diet containing 10(10) CFU live KB290, 10(10) CFU live KB392, 15 mg heat-killed KB290 or 600 μg crude extract of EPS-b for 1 day. Live KB290 (P < 0.01), heat-killed KB290 (P < 0.05) and crude EPS-b at 600 μg (P < 0.05) per mouse significantly enhanced cytotoxic activity; however, live KB392 had no effect. CONCLUSIONS Both live and heat-killed KB290 and crude EPS-b significantly enhanced cytotoxic activity of mouse splenocytes. SIGNIFICANCE AND IMPACT OF THE STUDY We demonstrated that EPS-b produced by KB290 has a critical role in enhancing cell-mediated cytotoxic activity in mouse spleen.

The content of stromal clonogenic cells in hemopoietic and lymphoid organs of mice and guinea pigs decreases with age. This drop is most pronounced in the thymus of mice and guinea pigs and in mouse spleen (more than 12-, 75-, and 8-fold, respectively). The contents of stromal clonogenic cells in the bone marrow of old mice and in the spleen of old guinea pigs are reduced by 50 and 40%, respectively, in comparison with young animals. These data indicate that the content of committed and inducible osteogenic precursors can decrease with age.

A hybridoma against the nucleoside m6A has been obtained from mouse spleen. This hybridoma was named H65 and it secretes monoclonal antibodies anti-m6A. The competition assays showed that the monoclonal antibody was highly specific for m6A nucleoside.

When mice were physically restrained in 50-ml tubes for 24 h, a marked decrease of NK activity was demonstrated in parallel with the elevation of serum corticosterone levels. The release of mice from restraint stress resulted in the recovery of NK activity, with a decrease of serum corticosterone levels within 48 h. Using this stress model, we also investigated the influence of restraint stress on mouse Th1/Th2 balance. Consistent with the decrease of NK activity, IFN-γ production of mouse spleen cells greatly reduced after suffering from restraint stress. In contrast, the IL-4 producing ability of spleen cells was not so much affected by restraint stress. These results initially indicated that stress may induce the skewing of the Th1/Th2 balance toward Th2-dominant immunity, which stimulates the occurrence of infectious diseases and allergic disorders.

Mouse spleen lymphocyte (SL) cells show a three to four-fold increase in [3H]-thymidine incorporation when incubated in tumour cell media, or in media containing tumour cell cytosol. Agarose gel chromatography of both [3H]-thymidine-labelled tumour cell media and cytosol shows a sharp peak of DNA-associated material eluting at about 60 kDa. This DNA-associated material is imported rapidly and efficiently by SL cells and is recoverable from their cytosol. The stimulating effect on SL cell thymidine incorporation resides primarily, if not exclusively, in this extruded/cytosolic 60 kDa DNA material. Tumour cells incubated in media containing normal or liver, but not tumour, cytosol show a reduced rate of [3H]-thymidine incorporation, indicating competition between normal and tumour associated DNA complexes. The results indicate that such cell-extruded DNA complexes may transmit 'genetic messages' to other cells, and are discussed in terms of interactions in the tumour-bearing host.

Aluminum hydroxide (alum) is the most widely used adjuvant in human vaccines. Nevertheless, it is virtually unknown whether alum acts on B cells. In the present study, we explored the direct effect of alum on Ig expression by murine B cells in vitro. LPS-activated mouse spleen B cells were cultured with alum, and the level of isotype-specific Ig secretion, IgG1 secreting cell numbers, and Ig germ-line transcripts (GLT) were measured using ELISA, ELISPOT, and RT-PCR, respectively. Alum consistently enhanced total IgG1 production, numbers of IgG1 secreting cells, and GLTγ1 expression. These results demonstrate that alum can directly cause IgG1 isotype switching leading to IgG1 production.

Naive and memory CD4 T cells from mouse spleen, alone or in a 1:1 mixture, were tested for Con A-induced proliferation in limiting dilution cultures. Dose–response curves for naive cells were linear, but curves for memory cells were hyperbolic, suggesting that positive responses required the activation of several cells of the memory type. Mixtures (1:1) gave zigzag curves, consistent with a previously described quantitative model in which memory cells block naive cell proliferation at low multiplicities and generate their own positive responses at higher multiplicities. Inhibition of naive cell proliferation by memory cells could be mimicked by IL-10 and blocked by anti-IL-10 antibody. IL-2 addition converted the multihit dose curves of memory T cells to single-hit curves, suggesting that poor IL-2 production limits growth in memory cell cultures. Surprisingly, IL-2 addition to cultures of naive cells led to a decrease in proliferation at high cell input doses. This inhibitory effect of IL-2 could be blocked by antibody to IL-10, and may reflect the presence of contaminating memory cells in the naive cell preparations. These models for analysis of interaction between naive and memory T cells in limiting dilution conditions point to a series of reciprocal interactions between IL-10 and IL-2 producing cells.