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Study was conducted on mouse spleen cells, cultured and incubated in-vitro with Holy basil and Thai basil, to observe their effect on proliferation. Four dilutions, namely 1:1, 1:5, 1:25, and 1:125...

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.

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.

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.

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).

Thymosin, composed of many peptides, is distributed in animal and has immunomodulating effects. However, many factors could affect its bioactivity. Chitosan microspheres containing pig thymosin (CMPT) were prepared through W/O emulsion cross-linking method. In this study, thymosin activity between pig-thymosin and CMPT was investigated through E-rosette test and mouse spleen lymphocyte proliferation (MPLP) experiment in vitro. Results showed that CMPT has the same immunomodulatory activity as pig thymosin. When pig-thymosin was 1.5 μg / ml, E-rosette binding rate was 63%, and E-rosette binding rate of CMPT reached 60%。MPLP rate was 73.3% and 68.5%, respectively. CMPT have the capacity to keep the E-rosette binding and MPLP activity of pig thymosin. This may help to improve traditional formulation and bioavailability of thymosin, and expand its scope of application in clinic.

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.

Zusammenfassung Wir untersuchten die biologischen Eigenschaften der verschiedenen Bordetella bronchiseptica-Stamme, die aus Schweinebestanden mit unterschiedlichem Krankheitsstatus (Atemwegserkrankungen) isoliert wurden. Unser Ziel war, das Exotoxinbildungsvermogen nachzuweisen, sowie die Exotoxin-bildung als biologischer Marker fur Bordetella bronchiseptica-Stamme und daruber hinaus die Eigenschaften des Toxins naher zu bestimmen. Aus den Ergebnissen sind folgende Schlusfolgerungen zu ziehen: 1. Exotoxinbildung und Virulenzgrad sind eng miteinander verbunden. Mit zunehmender Virulenz steigt auch die Exotoxinbildung an. 2. Subletale Dosen des reinen Exotoxins und toxischer Stamme bei i. v. Applikation rufen nach 7 Tagen eine schwere Atrophie der lymphatischen Organe hervor, was sich in einer Verkleinerung der Malpighischen Korperchen der Milz ausert. Gleichzeitig wird die B-Lymphozytenzahl im Blut verringert. Demgegenuber bewirken die nicht exotoxinbildende Stamme eine auffallige Hyperplasie der lymphatischen Organe und parallel dazu einen Anstieg der B-Lymphozyten. 3. In Anbetracht des engen Zusammenhanges zwischen Toxinbildung und Virulenz ist die Reaktion der Milz zugleich ein Ausdruck fur die exotoxinbildende Eigenschaft von B. bronchiseptica, aber zugleich auch fur den Virulenzgrad der B. bronchiseptica-Stamme. Deshalb kann die im Mause-Milztest nachgewiesene Exotoxinmenge als ein biologischer Marker der B. bronchiseptica-Stamme angesehen werden, wobei zu berucksichtigen ist, das auch geringe Unterschiede in der Exotoxinbildung der Stamme mit diesem Test exakt festgestellt werden konnen. Summary Epizootiological studies on porcine atrophic rhinitis. IV. Influence of heat-labile toxin of Bordetella bronchiseptica strains on lymphatic organs of mice The biological properties of various Bordetella bronchiseptica strains isolated from pig herds of varying disease status (respiratory disease) were studied. The objective was to demonstrate ability to produce exotoxin and also to study exotoxin formation as a biological marker for B. bronchiseptica strains and also to study the properties of the toxin. The results gave the following conclusions: 1. Exotoxin formation and degree of virulence were closely related. Increased virulence was associated with increased production of toxin. 2. Sublethal doses of pure exotoxin and toxic strains given i. v. produced after 7 days a severe atrophy of the lymphatic organs as shown by smaller spleen Malpighian bodies. At the same time there was reduction of the number of B-lymphocytes in the blood. In contrast, strains that did not produce exotoxin caused an increase in size of lymphoid organs and a parallel rise in B-lymphocytes. 3. In connection with this close relationship between toxin production and virulence, the reaction of the spleen is both an expression of the exotoxin-forming character of B. bronchiseptica and also of the grade of virulence of the organism. The mouse spleen test as an indicator of exotoxin amount can thus be used as a biological marker for B. bronchiseptica strains, bearing in mind that even small differences in toxin formation of strains can be precisely determined by this test. Resume Recherches epizootologiques sur a rhinite atrophique du porc IV. Influence d'une toxine thermolabile de souches de Bordetella bronchiseptica sur des organes lymphatiques de souris On a recherche les proprietes biologiques de differentes souches de Bordetella bronchiseptica, isolees dans des exploitations porcines au statuts de sante differents (maladies de l'appareil respiratoire). Le but fut de mettre en evidence la possibilite de formation d'exotoxine et de determiner cette formation comme marqueur biologique pour des souches de Bordetella bronchiseptica et de definir en plus les proprietes de la toxine. Les conclusions suivantes ont ete titrees a partir des resultats: 1. La formation d'exotoxine et le degre de virulence sont etroitement lies. La formation d'exotoxine augmente avec la virulence. 2. Des doses subletales de l'exotoxine pure et de souches toxiques par application i/v provoquerent une grave atrophie des organes lymphatiques apres 7 jours qui s'est manifestee par une atrophie des corpuscules de Malpighi dans la rate. Le nombre des lymphocytes B a en meme temps diminue dans le sang. Les souches ne formant pas de toxine ont provoque par contre une hyperplasie manifeste des organes lymphatiques et une augmentation parallele des lymphocytes B. 3. En considerant l'etroite correlation entre la formation de toxine et la virulence, la reaction de la rate et l'expression en meme temps de la propriete de formation d'exotoxine de B. bronchiseptica et du degre de virulence des souches de B. bronchiseptica. Le test de la rate chez des souris pour la mise en evidence quantitative d'exotoxine peut donc etre considere comme un marqueur biologique des souches de B. bronchiseptica, tout en considerant que de faibles differences dans la formation d'exotoxine des souches peuvent etre mises exactement en evidence avec ce test. Resumen Estudios epizootologicos sobre la rinitis atrofica del cerdo IV. El influjo de la toxina termolabil de las estirpes Bordetella bronchiseptica sobre los organos linfaticos en los ratones Examinamos las propiedades biologicas de diversas estirpes de Bordetella bronchiseptica, aisladas de efectivos porcinos con estado desigual de salud (enfermedades de las vias respiratorias). El fin que nos proponiamos era poner en evidencia la facultad de formacion de exotoxina como marcador biologico para las estirpes Bordetella bronchiseptica y definir mas alla, en detalle, las propiedades de la toxina. De los resultados obtenidos se sacan las conclusiones siguientes: 1. La exotoxinogenia y el grado de virulencia se hallan relacionados estrechamente entre si. Con la virulencia creciente tambien aumenta la formacion de exotoxina. 2. Dosis subletales de exotoxina pura y de cepas toxicas provocan tras la aplicacion iv., al cabo de 7 dias, una atrofia grave de los organos linfaticos, lo cual se manifiesta por una achicadura de los corpusculos de Malpigio en el bazo. Al mismo tiempo disminuye el numero de linfocitos B en sangre. Por el contrario, las estirpes que no son exotoxinogenas causan una hiperplasia ***llamativa des los organos linfaticos y el aumento paralelo de linfocitos B. 3. En consideracion a la relacion estrecha entre la toxinogenia y la virulencia, la reaccion del bazo es al mismo tiempo la expresion de la propiedad exotoxinogena de B. bronchiseptica y un indicio del grado de virulencia de las estirpes de B.- bronchiseptica. Por lo tanto, la cantidad de exotoxina puesta en evidencia en la prueba del bazo de raton puedo considerarse como un marcador biologico de las cepas de B. bronchiseptica. Con esta prueba mencionada tambien se pueden poner en evidencia, con toda exactitud, diferencias incluso escasas en la exotoxinogenia de las estirpes.

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.

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.

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.

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.

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

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...

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.

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.

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.

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.

In the present study the role of tumor necrosis factor α (TNFα) in endotoxin-induced mitogenicity of mouse spleen cells was examined. TNFα was found to enhance endotoxin-induced proliferation of spleen cells. However, in the absence of endotoxin, tumor necrosis factor treatment was without effect. An antiserum against TNFα completely abolished the mitogenic effect of endotoxin in spleen cell cultures. This inhibition was reversed by exogenously added TNFα. The TNFα production in spleen cell cultures reached a maximum after 6 h. When the antiserum was added to cultures at various time intervals following endotoxin addition, maximal inhibition was observed at a time point 6 h after the start. These data suggest that TNFα has a function in endotoxin-induced mitogenicity of murine spleen cells.

The International Standard (IS) for Recombinant DNA-Derived (rDNA) Erythropoietin (EPO) (in ampoules coded 87/684) and three other rDNA EPO preparations in ampoules coded 87/690, 87/696 and 88/574 respectively, were compared with two preparations of highly purified human urinary (HU) EPO and the 2nd International Reference Preparation of Human Urinary Erythropoietin for Bioassay (2nd IRP) by 26 laboratories in 11 countries using a wide range of in-vivo and in-vitro bioassays and immunoassays. These EPO preparations were also compared by electrophoresis and isoelectric focusing. Estimates of EPO content in terms of the 2nd IRP by all in-vivo bioassay methods gave combined unweighted geometric means (with 95% fiducial limits) of: 86 (75–99) IU/ampoule for the IS, 81 (70–94) IU/ampoule for 87/690, 58 (48–71) IU/ampoule for 87/696 and 120 (100–143) IU/ampoule for 88/574. Mean estimates of EPO content in terms of the 2nd IRP by in-vitro bioassays (except receptor assays) were larger than, and those by immunoassays were similar to, the mean estimates by in-vivo bioassays. The use of purified rDNA or HU EPO as standards in place of the 2nd IRP reduced the inter-laboratory variability of estimates of purified EPO preparations by in-vivo and in-vitro bioassays and by immunoassays, and reduced the variability of overall mean estimates for each of these preparations between the three types of method. The inter-laboratory variability of immunoassay estimates of human serum EPO was similar whether the 2nd IRP or one of the purified EPOs was used as standard. Significant differences in in-vivo and in-vitro biological, immunological and physicochemical properties were found between these four rDNA EPO preparations and between them and the HU EPO in the two purified preparations and in the 2nd IRP. There were also differences between the immunoreactivities of the two serum EPO samples included in the study, and between them and the immunoreactivities of the purified EPOs. The differences between rDNA EPOs appeared to be related to differences between the cells used for their biosynthesis, but may also be the result of differences in purification methods and of inter-batch variations. Significant differences in assay specificity were observed within each of the three general types of method. The specificity of the in-vivo bioassays was influenced by the route of hormone administration. The specificities of the mouse spleen cell in-vitro bioassays differed from that of the mouse spleen receptor-binding assay. The specificity of one-site immunoassays differed with the type of EPO used as antigen or tracer, with most notable differences between assays using antisera to rDNA and HU EPO. Two-site immunoassays gave significantly lower estimates for serum EPO than one-site immunoassays. On the basis of these results, the World Health Organization (WHO) Expert Committee on Biological Standardization established the preparation in ampoules coded 87/684 as the International Standard for Recombinant DNA-Derived Erythropoietin with an activity of 86 IU Erythropoietin, rDNA-Derived, per ampoule. It also recommended that the WHO keep under consideration the establishment of separate standards for naturally occurring EPO and for rDNA EPO produced in different cell lines. Journal of Endocrinology (1992) 134, 459–484

The expression of the beta-endorphin receptor on both activated and unstimulated mouse spleen cells was studied. Results showed that unstimulated cells have only one type of beta-endorphin receptor with a specific low affinity (Kd = 1.034 +/- 0.0237 x 10(-7) M, 25,000 sites/cell). After Con A stimulation, cells express two types of receptors, one with a low affinity (Kd = 1.034 +/- 0.024 x 10(-7) M, 320,000 sites/cell) and the other with a high affinity (Kd = 1.052 +/- 0.033 x 10(-9) M, 49,000 sites/cell). The kinetic experiments during 4 days after Con A activation indicated that the receptor of high affinity emerged from 24 to 72 h, while the low affinity one increased in number after stimulation. The receptor numbers of both high and low affinity ones reached a maximum peak at 72 h, then began to decline. The addition of exogenous rIL-2 depressed the Con A-induced increment of the receptor numbers of both the high and low affinity ones, but enhanced the proliferative response of the cells. It is suggested that the degree of the expression of the receptors does not simply depend on the mitogenic degree of the cells. In addition, our experiment demonstrated that splenocytes cultured in medium with or without Con A or Con A + rIL-2 for 96 h did not secrete any detectable amount of beta-endorphin with use of the RIA assay, which is sensitive enough to detect the much lower levels of beta-endorphin than that necessary for biological effects. We suggest that the expression of the high affinity beta-endorphin receptor on the activated T-lymphocytes may have to precede the production of IL-2 to potentiate the T-cell proliferative response. The mechanisms and modes of interaction between the neuroendocrine system and the immune system were discussed.

The relative persistence capacity in mouse spleen of 10 and 9 BCG substrains from liquid and dried vaccines, respectively, was evaluated in two studies. Recoverable BCG colony counts from mouse spleen were determined at given days on solid medium in the two studies during a period of 1–360 and 1–345 days, respectively, after the intravenous BCG vaccination, performed with two different viable units. From 36 000 (study 1) and 21 600 (study 2) recoverable BCG colony counts, 180 and 108 mean relative persistence capacity values were estimated to test the residual virulence during the follow-up time, using computerized statistical analysis. The early and late trends of mean relative persistence capacity of the BCG substrains in mouse spleen were tested by linear regression analysis and analysis of variance and covariance; then with ranked adjusted group mean relative persistence capacity, Gabriel's simultaneous test procedure was performed for multiple comparison to diminish type 1 error in statistical inference and in objective interpretation of the experimental results. The associations of the ranked mean relative persistence capacity of the BCG substrains at the different sacrifice days of mice were also analyzed by Kendall's test of concordance. The early, late, and overall relative persistence capacity reflects the residual virulence of the BCG substrains and provides information on the required protective efficacy (immunogenicity) and adverse reactions (reactogenicity), allowing the appropriate vaccination dose, expressed in viable units of the substrain used, to be determined. Key words: BCG substrains, residual virulence, relative persistence in mouse spleen, multiple comparison.

We found that a novel and potent immunoglobulin production stimulating factor (IPSF) was contained in soybean trypsin inhibitor (STI) preparation. The IPSF significantly enhanced IgM production by mouse spleen lymphocytes. To clarify the mechanism of the IPSF activity, the effect of the IPSF on the expression of some cytokines was examined. The IPSF promoted the expression of interleukin-6 (IL-6) and IL-10 mRNA. The IgM production stimulating activity was partially but significantly suppressed by IL-6 neutralization antibody. These data suggest that the IPSF stimulates IgM production, at least partially, via the enhancement of IL-6 production. To reveal the IPSF is not STI, STI preparation was fractionized by anion-exchange chromatography and the IPSF activity of each fraction was analyzed. Active fraction was eluted distinct fraction containing STI. Active fraction contained some proteins whose molecular weights were about 29 kDa. These suggest that IPSF in STI preparation is not STI itself but contaminants. Taken together, the about 29 kDa protein and/or its complexes with several proteins exist in STI preparation could have a potent IPSF activity and the activity was exerted, at least partially, via stimulation of IL-6 production.

A monoclonal antibody against 4-aminobenzoate hydroxylase (EC 1.14.13.27) from Agaricus bisporus, a common edible mushroom, has been produced by the fusion of BALB/c mouse spleen cells immunized with the denatured enzyme and P3x63Ag8U1 myeloma cells in order to locate and characterize the catalytic site of the enzyme. The monoclonal antibody immunoblotted the enzyme and immunoprecipitated its apoenzyme. The immunoprecipitation was inhibited in the presence of FAD, and the monoclonal antibody competitively inhibited the binding of FAD to the apoenzyme. The monoclonal antibody, therefore, recognizes the FAD-binding site of 4-aminobenzoate hydroxylase. Interestingly, it was shown that the monoclonal antibody was cross-reactive with FAD-dependent enzymes such as salicylate hydroxylase (EC 1.14.13.1) and D-amino acid oxidase (EC 1.4.3.3), and that it was specific for the FAD-binding sites of these enzymes. This fact suggests that these FAD-dependent enzymes have immunologically similar structures on their FAD-binding sites.

Formalin-fixed, paraffin-embedded (FFPE) samples are commonly used for archiving pathology samples for use by many researchers including clinical research labs. These samples provide a valuable tool for retrospective studies of diseases such as cancer. Traditional methods for the purification of DNA or RNA from FFPE tissue samples are often labor intensive, include the use of hazardous organic reagents, and involve difficult pre-processing steps. Here, we describe development of automated methods for the purification of RNA or DNA from FFPE tissue sections using the Maxwell® RSC Instrument, which can process between 1 and 16 samples. DNA was purified from FFPE mouse brain, liver, lung, skin and spleen as well as FFPE human colon. RNA was purified from FFPE mouse brain, liver, lung, skin and spleen as well as FFPE human breast tissue. Eluates were tested for inhibition in qPCR or RT-qPCR. A comparison with another automated method was performed with FFPE mouse spleen and skin and a human tissue. The Maxwell RSC methods produced high quality amplifiable DNA or RNA from a variety of sample types with little or no inhibition. In addition, these methods simplified pre-processing, minimized hands-on time, and did not use hazardous organics. Citation Format: Michelle Mandrekar, Douglas Horejsh, Samantha Lewis, Chris Moreland, Marjeta Urh. Automated RNA and DNA purification from FFPE samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4846. doi:10.1158/1538-7445.AM2015-4846

Major analytical challenges encountered by shotgun proteome analysis include both the diversity and dynamic range of protein expression. Often new instrumentation can provide breakthroughs in areas where other analytical improvements have not been successful. In the current study, we utilized new instrumentation (LTQ FT) to characterize complex protein samples by shotgun proteomics. Proteomic analyses were performed on murine spleen tissue separated by magnetic beads into distinct CD45 and CD45 cell populations. Using shotgun protein analysis we identified 2,000 proteins per cell group by over 12,000 peptides with mass deviations of less than 4.5 ppm. Datasets obtained by LTQ FT analysis provided a significant increase in the number of proteins identified and greater confidence in those identifications and improved reproducibility in replicate analyses. Because CD45 and not CD45 cells are able to regenerate functional pancreatic islet cells in a mouse model of type I diabetes, protein expression was further compared by a subtractive proteomic approach in search of an exclusive protein expression profile in CD45 cells. Characterization of the proteins exclusively identified in CD45 cells was performed using gene ontology terms via the Javascript GoMiner. The CD45 cell subset readily revealed proteins involved in development, suggesting the persistence of a fetal stem cell in an adult animal. Molecular & Cellular Proteomics 4:1459–1470, 2005. The use of proteomic technologies for global characterization of proteins expressed in cells, tissues, and biological fluids is a key component in furthering our ability to understand biological processes in normal and diseased states. In many proteomic studies, cells or tissues are characterized by profiling and comparing proteins expressed in treated and untreated cells, cancerous versus normal tissues, or various cell populations. A seminal tool in this development has been the application of mass spectrometry technologies to identify proteins and post-translation modifications in complex protein mixtures. The term “shotgun proteomics” is used to de

In this study, we have investigated the expression of cytokine mRNAs in mice cutaneously exposed to formaldehyde using semiquantitative RT-PCR. We show that formaldehyde induced the long-lasting expression of IL-4 and IFN-gamma mRNAs and the transient expression of IL-13 mRNA in mouse spleen and draining lymph nodes. The transient increases in IL-2, IL-15, IL-12p40, IL-15 and IL-18 mRNAs, but long-lasting IL-15 mRNA were only seen in the formaldehyde-exposed mouse spleen. Moreover, a weak contact hypersensitivity (CH) and the significant increases in IL-4 and IFN-gamma mRNAs were detected in the ear skin of formaldehyde-cutaneously exposed mice when rechallenged mouse ears. Furthermore, CH as measured by mouse ear swelling response was positively correlated with IL-4 and IFN-gamma mRNA levels in the challenged ears. This study thus suggests that the induction of Th1 and Th2 cytokine mRNAs, particularly IL-4 and IFN-gamma, are a common immunological feature caused by contact allergens irrespective of strong or weak contact allergens. The analysis of IL-4 and IFN-gamma mRNAs may be useful markers in establishing the novel test for predicting chemical sensitizing potentials.

We have designed a two-phagemid system for the construction of very large non-phage displayed Fab antibody libraries in E. coli approaching 1012 members. The system can accommodate both periplasmic and cytoplasmic Fab expression and should prove useful for the direct selection of functional antibodies by genetic techniques. We successfully alleviate problems of Fab vector instability and report a set of improved 5′ primers for the amplification of mouse Ig VH repertoires from mouse spleen. These primers have no more than one mismatch in the last 11 bases for >95% of mouse Ig VH genes and minimize the amount of N-terminal amino acid changes while maintaining the flexibility of periplasmic or cytoplasmic antibody expression in E. coli.

The effects of low dose-rate γ-irradiation on the induction of DNA damage were investigated in the spleen of C57BL/6N mice. A modified single-cell gel electrophoresis (comet assay) was employed to measure DNA damage. Mice were irradiated with 137Csγ-rays at 1.2 mGy/h for 23 days (0.5 Gy total). The amount of DNA damage was compared to that in the mice irradiated with the same dose of X-rays at 96 Gy/h (high dose-rate). When the mice irradiated at the low dose-rate were then exposed to 1 Gy of X-rays, the initial amount of DNA damage was less than that in mice, which received the 1 Gy only. These results indicated that damage was repaired during irradiation period and that the low dose-rate irradiation induced an adaptive response in terms of DNA damage

This protocol describes a procedure for obtaining a single-cell suspension from mouse spleen or solid tissue. Once a single-cell suspension is prepared, multicolor flow cytometry is used to identify subsets of immune cells in the samples of interest. This protocol can be used to evaluate the following immune cell populations, defined by the expression of certain cell surface molecules: total leukocytes, T lymphocytes and CD4+ and CD8+ T-cell subsets, B lymphocytes, natural killer cells, dendritic cells, and immature myeloid cells.

In this study the effect of PDT on the antitumoral specific immunologic response was evaluated. We compared the specific cytolytic activity (CLA) by a chromium release assay of primed mouse spleen T lymphocytes sensitized against syngeneic mastocytoma P511 cells. P511 cells, or lymphocytes, or both cells were treated or not with photofrin and/or light (514 nm). Photofrin II alone (1 (mu) g/ml, 2 hours) reduced CLA 59% when P511 were treated. Photofrin II (1 (mu) g/ml) followed by light (25 Joules/sq cm) also reduced CLA 35%. Photofrin II alone (0.5 (mu) g/ml, 2 hours) reduced CLA 8% when only lymphocytes were treated. And Photofrin II (0.5 (mu) g/ml) followed by light (25 Joules/sq cm) also reduced CLA 45%. When both cells were treated with Photofrin II alone or followed by light (25 Joules/sq cm) the CLA was also reduced respectively 19, 41%.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Preparations of murine recombinant interferon (Mu-rIFN)-alpha, -beta and -gamma were assessed for their influence on in vitro growth of mast cells from normal mouse spleen cells (Sp C). Mast cell growth was inhibited by Mu-rIFNs when Sp C were exposed throughout the entire culture period to Mu-rIFNs. The most potent inhibitor of mast cell growth was Mu-rIFN-gamma, followed by Mu-rIFN-beta; Mu-rlFN-alpha had little effect. When added to IC-2 cells, clonal mast cell progenitor, both Mu-rlFN-beta and -gamma), significantly inhibited proliferative response of the target cells. The suppressive effect of Mu-rIFNs on IC-2 cells was selectively abolished by monoclonal antibodies against Mu-rIFN-beta and -gamma.

Six murine monoclonal antibodies (MAbs) against staphylococcal enterotoxin A (SEA) and enterotoxin E (SEE) were prepared by fusion of myeloma cells with mouse spleen cells immunized with SEA and SEE. Of five MAbs to SEA tested, two MAbs were reactive with only SEA, whereas three were specific for both SEA and SEE. On the other hand, one MAb to SEE was found to be specific for only SEE. To study specificities of the combining sites of these MAbs, competitive binding assays with either SEA or SEE and horseradish peroxidase conjugated MAbs were performed using unconjugated MAbs as inhibitors. The results obtained in the assays suggest that different epitopes may be located on SEA and that some of them may be cross-reacting epitopes between SEA and SEE. Key words: enterotoxins, monoclonal antibodies, Staphylococcus aureus.

To the Editor: We read with interest that a second thrombin receptor, designated protease activated receptor 3 (PAR3), has been cloned from human and mouse tissues.[1][1] The human and mouse PAR3 cDNAs were cloned by screening a human small intestinal cDNA library and a mouse spleen cDNA library

The rate of clearance of Salmonella typhimurium from the mouse spleen is under H-2 linked genetic control. The results of the present study, with H-2 recombinant mice on a C57BL/10 background, suggest the involvement of at least two loci, one in the D region and the other in the K-A alpha chromosomal segment.