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Aims: Silymarin (SIL), a mixture of phenolic compounds, has a pleiotropic mode of action on various cell types, including immune cells. In this study, we investigated the concentration-dependent effect of SIL on proliferation of concanavalin A (CoA)-stimulated mouse spleen T lymphocytes, their viability, and secretion of IFN-g and IL-4 cytokinesex vivoin relation to gene expressions of transcription factors nuclear factor kappa B and Foxp3. In addition, metabolic activity of T cells was determined as changes in the mitochondrial membrane potential and apoptosis.Material/Methods: Isolated splenocytes were stimulated with lectin CoA and treated with SIL atthe concentrations of 5, 10, 20, and 40 μg/ml for 70 h and unstimulated cells served as the control. Cultures of splenocytes were evaluated for proliferation index following BrdU incorporation and viability of cells after trypan blue staining. Gene expressions of transcription factors and cytokines were assessed using real-time PCR, whereas ELISA test was applied to measure cytokine secretion. Mitochondrial membrane potential and apoptosis were determined by flow cytometry.Results: We demonstrated that CoA-activated mouse spleen T lymphocytes show different susceptibilities to low (£10 μg/ml) and higher (20 and 40 μg/ml) SIL concentrations. Low concentrations resulted in increased proliferation, cytokine secretion, and mitochondrial membrane potential and reduced transition of cells to apoptosis. High concentration of SIL had the opposite effect without exerting significant cytotoxicity and upregulated genes for cytokines and transcription factors on mRNA level. It is possible that individual subpopulations of T cells induced by CoA were differentially affected by the various SIL concentrations and the dose of 40 μg/ml had the profound suppressive effect. This correlated with the highest expression of Foxp3 factor, indicating that this dose stimulated preferential differentiation to Tregs lymphocytes.Conclusions: Treatment with suitable doses of SIL can provide potential benefits in the modulation of host immune functions in various diseases.

Anti-CD25 antibodies were immobilized on polypropylene (PP) nonwoven fabrics to specifically remove mouse regulatory T cells (Tregs) from mouse spleen cells. PP fibers were coated with peptide nanosheets, which were prepared by self-assembling of a mixture of X-poly(sarcosine)-b-(l-Leu-Aib)6 (X: glycolic acid or a phenylboronic acid) and Y-poly(sarcosine)-b-(d-Leu-Aib)6 (Y: glycolic acid or diazirine derivative). Anti-CD25 antibodies were immobilized by covalent linking between the sugar moiety of the antibody and the phenylboronic acid group on the peptide nanosheet. The removal rate of mouse Tregs from the mouse spleen cells was more than 95% only by passing the filters, while the nonspecific removal rates of other cells were less than 15%. The coating of peptide nanosheets on PP fibers was very effective to provide a suitable environment for the immobilized antibody to interact with the counterpart cells while the coating suppressed nonspecific adsorption of other cells.

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.

This paper introduces a configurable combined laser, ultrasound, and elasticity (CLUE) imaging platform. The CLUE platform enables imaging sequences capable of simultaneously providing quantitative acoustic, optical, and mechanical contrast for comprehensive diagnosis and monitoring of complex diseases, such as cancer. The CLUE imaging platform was developed on a Verasonics ultrasound scanner integrated with a pulsed laser, and it was designed to be modular and scalable to allow researchers to create their own specific imaging sequences efficiently. The CLUE imaging platform and sequence were demonstrated in a tissue-mimicking phantom containing a stiff inclusion labeled with optically-activated nanodroplets and in an ex vivo mouse spleen. We have shown that CLUE imaging can simultaneously capture multi-functional imaging signals providing quantitative information on tissue.

Summary This protocol describes the comparative proteomic profiling of the spleen of wild type versus mdx-4cv mouse, a model of dystrophinopathy. We detail sample preparation for bottom-up proteomic mass spectrometry experiments, including homogenization of tissue, protein concentration measurements, protein digestion, and removal of interfering chemicals. We then describe the steps for mass spectrometric analysis and bioinformatic evaluation. For complete details on the use and execution of this protocol, please refer to Dowling et al. (2020)., Graphical Abstract, Highlights • Mass spectrometry-based proteomic analyses of spleen tissue samples • Comparative profiling of the spleen from the mdx-4cv model of dystrophinopathy • Protein extraction and protein digestion for peptide mass spectrometric analysis • Bioinformatic analysis of large proteomic datasets for comparative studies, This protocol describes the comparative proteomic profiling of the spleen of wild type versus mdx-4cv mouse, a model of dystrophinopathy. We detail sample preparation for bottom-up proteomic mass spectrometry experiments, including homogenization of tissue, protein concentration measurements, protein digestion, and removal of interfering chemicals. We then describe the steps for mass spectrometric analysis and bioinformatic evaluation.

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.

Two sulfated polysaccharides SCP3 and SCP5 with degree of substitution (DS) 0.45 and 0.13 were obtained by modifying the Cyclocarya paliurus polysaccharide (CP) with chlorosulfonic acid-pyridine method. Results demonstrated that the physicochemical properties, molar ratios of monosaccharide composition, molecular weights, and in vitro immunomodulatory activity were changed after being sulfated. Splenocytes proliferation assay showed that both CP and sulfated polysaccharides have immunomodulatory, while sulfated polysaccharides were stronger. CP mainly stimulate the production of the Th1-type cytokines, SCP3 and SCP5 not only significantly enhanced the secretion level of Th1-type cytokines, but also promote the Th2-type cytokines. Sulfated polysaccharides could significantly stimulate splenic lymphocyte proliferation synergistically with ConA or LPS. SCP3 significantly promoted the proliferation of T lymphocytes, while SCP5 stimulated the proliferation of B lymphocytes, which might be related to the DS. These results indicated that sulfation could improve the immune-enhancing effects of CP and different DS has different immunomodulatory activity.

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.

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.

Ultrasound-switchable fluorescence (USF) imaging was recently developed to overcome the limitation of the poor spatial resolution of the conventional fluorescence imaging in centimeter-deep tissue. However, in vivo USF imaging has not been achieved so far because of the lack of stable near-infrared contrast agents in a biological environment and the lack of data about their biodistributions. In this study, for the first time, we achieved in vivo USF imaging successfully in mice with high resolution. USF imaging in porcine heart tissue and mouse breast tumor via local injections were studied and demonstrated. In vivo and ex vivo USF imaging of the mouse spleen via intravenous injections was also successfully achieved. The results showed that the USF contrast agent adopted in this study was very stable in a biological environment, and it was mainly accumulated into the spleen of the mice. All the results of USF imaging were validated by a commercial micro-CT., Comment: 14 pages, 6 figures

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.

The spleen is an important organ of the immune system responsible for filtering blood and initiating immune responses to blood-borne antigens. Various blood, lymphoid, and hematopoietic cell types may be isolated from spleen samples for further study of the immune system. Preparing a true single cell suspension of the primary tissue sample will optimize cell separation by avoiding additional cell loss and enabling maximum labeling of the target cells. This protocol describes how to harvest cells from a spleen sample, and prepare a single cell suspension prior to performing cell isolation.

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.

In this study, we revealed that a Mekabu (Udaria pinnantifida) extract enhanced immunoglobulin (Ig) production of mouse spleen lymphocytes. Furthermore, it was suggested that water-soluble and high molecular weight ingredients in the Mekabu extract have significant enhancing effect on Ig production. Therefore, fucoidan was estimated as the active component.

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.

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).

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.

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/106 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.

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.

This work introduces minimum accumulative degeneracy, a variant of the degenerate primer design problem, which is particularly useful when a large number of sequences are to be covered by a set of restricted number of primers. A primer set, which is designed on a minimum accumulative degeneracy basis, especially helps to reduce nonspecific PCR amplification of undesired DNA fragments, as fewer primer species are present in PCR. A Boltzmann machine is designed to solve the minimum accumulative degeneracy degenerate primer design problem, called the MAD-DPD Boltzmann machine. This algorithm shows great flexibility, as it can be determined either to solve the problem with strict fidelity to covering all input sequences or to exclude some input sequences if it results in less degenerate primers. This Boltzmann machine is successfully implemented in designing a new set of primers for amplification of antibody variable fragments from mouse spleen cells, which theoretically covers more diverse antibody sequences than currently available primers. The MAD-DPD Boltzmann machine is available online at bioinf.cs.ipm.ir/download/MAD_DPD08172007.zip.

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.

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.

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.

マウス脾臓リンパ球の増殖および抗体産生に及ぼすカゼイン,ホエータンパク質,大豆タンパク質,小麦グルテンおよびペプチドの影響について検討した.牛β-カゼインは,マウス脾臓リンパ球のIgA,IgGおよびIgM産生を添加用量依存的に増強した.また,カゼイン由来ペプチドはIgA産生を増強し,小麦グルテン由来ペプチドはIgA,IgGおよびIgM産生を増強した.一方,ホエー,大豆タンパク質およびペプチドには脾臓リンパ球に対する抗体産生増強効果は認められなかった.また,β-カゼイン刺激により脾臓リンパ球における抗体産生細胞数の割合の増加が認められた.したがって,β-カゼインの抗体産生増強効果の発現には抗体産生細胞数の増加が関与することが示唆された.しかし,カゼイン由来ペプチド刺激による抗体産生細胞数の変化は認められず,カゼイン由来ペプチドのIgA産生増強効果には抗体産生細胞数の変化は関与していないことが示唆された.これらの結果より,カゼインおよび小麦グルテン由来ペプチドは生体内での免疫調節機能が期待され,免疫調節食品素材として利用できると思われる.

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.

The present study was carried out to evaluate the cytogenetic effect of manganese sulfate (MnSO4) in cultured mouse spleen cells and the possible protective effect of folic acid (FA) and/or vitamin B12 (VB12) on cell cultures treated with MnSO4. Cultured mouse spleen cells were treated with MnSO4 at 4 concentrations (10-6, 5×10-6, 10-5, 10-4). FA at 10-5, 5×10-5 and 10-4M, and VB12 at 5×10-6, 10-5 and 5×10-5 M were used for testing the protective effect on chromosome damage induced by MnSO4. The highest dose (10-4 M) of MnSO4 were treated simultaneously with FA and/or VB12. The cultures were set up for 24 h. The results indicate that MnSO4 alone induced a concentration-related increase in the percentage of chromosome aberrations. Both FA and VB12 reduced the percentage of aberrant cells in a significant (p

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.

Apoptotic leukocytes undergo cellular changes that are used as markers for “early” versus “late” stages of apoptosis. To ascertain if the order for acquisition of these changes is unique to specific hematopoietic cell types, we compared four leukocyte cell types and the following five apoptotic characteristics: MC540 incorporation, annexin V-FITC binding, propidium iodide (PI) labeling of hypodiploid nuclei, DNA fragmentation by a colorimetric assay, and cell membrane permeability to PI. The order for acquisition of these apoptotic characteristics was significantly different for each of the leukocyte cell types and for the mode of induction of apoptosis. It is interesting that the nuclear changes but not the membrane changes studied in mouse spleen cells required caspase activity. In summary, the acquisition of these apoptotic characteristics occurs through caspase-dependent and caspase-independent mechanisms, and importantly, the order for acquisition of the characteristics is specific for the cell type and for the mode of induction of apoptosis.

In this study we compare oxygen tension (Po 2) histograms measured with O2 microelectrodes and a new optical Po 2 measurement device, the OxyLite, in normal tissues (mouse spleen and thymus) and in tumors (R3230Ac in rats) ( n = 5–6). The transient response to glucose infusion or 100% O2 breathing (hyperoxia) was also measured in tumors. Po 2 histograms of spleen and thymus with the two devices were not different. The OxyLite tumor Po 2 histogram, however, was left-shifted compared with the microelectrode (median Po 21.0 vs. 4.0 mmHg, P = 0.016). Both probes responded to acute hyperglycemia with a mean increase of 3–6 mmHg, but the microelectrode change was not significant. The OxyLite consistently recorded large Po 2 increases (∼28 mmHg) with hyperoxia, whereas the microelectrode response was variable. The OxyLite averages Po 2 over an area that contains interstitial and vascular components, whereas the microelectrode measures a more local Po 2. This study demonstrates the importance of considering the features of the measurement device when studying tissues with heterogeneous Po 2 distributions (e.g., tumors).

Effects of bovine beta-casein (1-28) having a phosphoserine-rich region (Glu14-SerP-Leu-SerP-SerP-SerP-Glu-Glu21) and its chemically synthesized partial fragments on proliferation of lymphocytes and immunoglobulin production were investigated in mouse spleen cell cultures. The parent fragment 1-28 and all fragments containing SerP-Leu-SerP and/or SerP-SerP-SerP had a significant mitogenic effect, stimulated proliferation of lymphocytes induced by lipopolysaccharide, phytohemagglutinin, or concanavalin A, and increased immunoglobulin (IgG + IgM + IgA) or IgA levels in the cell cultures. In contrast, dephosphorylated beta-casein (14-21) and SerP-SerP amide had hardly any immunoregulatory activity. On the other hand, SerP-Leu-SerP amide reacted little with antibodies specific to bovine beta-casein (1-28), but beta-casein (14-21), and SerP-SerP-SerP amide obviously reacted with the antibody. These results confirm that the immunoregulatory activity of casein phosphopeptides is attributable to SerP-X-SerP, which may well be available as a non-allergic food ingredient having an adjuvant activity for mucosal IgA responses.

Item does not contain fulltext In this issue of Immunity, Luber et al. (2010) report a comprehensive quantitative proteome of in vivo mouse spleen dendritic cell (DC) subsets: a data set of encyclopedic value already revealing that DC subsets exploit different RNA sensors for virus recognition.

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.

The plaque assay years at Pittsburgh that included efforts to optimize the detection for single immunoglobulin M (IgM) and IgG antibody-producing cells are described. Sensitivity of the assay and precision of the plaque count was established, and a series of response curves were obtained that enabled to calculate the frequency of precursors and to identify the target of the antigen. If one ignored a number of anomalies of the curves and made the simple assumption that a single hit by antigen on a pre-committed precursor yielded a clone of antibody-forming cells, estimates of some 700 target cells (per mouse spleen) were obtained, figures not too far from the number that Ivan would later find. Years at the Paul Ehrlich Institute and visits to the Basel Institute for Immunology are described.

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.