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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...

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 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.

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.

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.

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.

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

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.

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.

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

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

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.

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

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

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.

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.

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.

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

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.

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

The term ‘dendritic cells’ (DCs) was introduced in 1973 by a team led by Ralph Steinman, future Nobel Prize winner. During ongoing research, this group found a new population of leukocytes in the mouse spleen. These cells were characterized by long cytoplasmic protrusions and had the ability to stimulate lymphocytes. Subsequent studies have shown that DCs are a group of professional APCs, and among this group are the most potent immuno stimulatory cells. DCs form a very hetero geneous popu

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.

This month's cover depicts, by virtue of the scales, the balance of experts' opinions that are provided in the featured Macrophage Viewpoint series. Superimposed on the scales are cartoon drawings of the different macrophage subtypes (classical, alternative and regulatory) discussed by Fleming and Mosser (pp. 2498-2502); the background is kindly provided by Joan Stein-Streilein and is reprinted with permission from Faunce, D. E. et al., J. Immunol. 2001. 166: 313-321 (© 2001 The American Association of Immunologists, Inc.). The background shows migrating F4/80(+) APCs (pink) forming clusters with NKT and B cells in the marginal zone of mouse spleen, in the ACAID model of peripheral tolerance. The F4/80 antigen is discussed in the Viewpoint by Siamon Gordon et al. (pp. 2472-2476), and the paper first describing the F4/80 antibody features as one of the EJI classics on the EJI homepage (www.eji-journal.eu)

This issue's cover image is provided by Chen et al., the authors of “A regulatory role for macrophage class A scavenger receptors in TLR4-mediated LPS responses” (pp. 1451–1460). The image depicts immunofluorescent staining of mouse spleen frozen sections and demonstrates that FITC-conjugated heat-killed E. coli are efficiently captured by spleen marginal zone macrophages – a process that is facilitated by MARCO expressed on macrophages.

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.

The first trial in the developmental phase of the "Performance Evaluation Program" based on the new programs of the International Council for Laboratory Animal Science (ICLAS) was carried out. ICLAS supplied test samples to each diagnostic laboratory for self-assessment of microbiological monitoring methods. We found that 1 mouse serum sample was positive for mouse minute virus and another was positive for Mycoplasma pulmonis antibodies, and 1 rat serum sample was positive for Sendai virus antibody. Mouse parvovirus was detected from mouse spleen and mesenteric lymph node homogenate, and Helicobacter spp. were detected from mouse feces. Corynebacterium bovis was isolated from a mouse skin sample. These results were in agreement with those notified by the ICLAS after the trial. After this trial, the program will eventually be made available to all diagnostic laboratories willing to participate in it.

During the primary culture of spleen fragments of newborn mice, a spherical body (d = circa 200 to 300 microns) as a three-dimensional cellular organization was formed. Continued production of blood cells from the spherical body was observed without changing its size for about 2 months of culture. Without growth factor, the spherical bodies produced mainly lymphocytes and macrophages. Addition of interleukin-3 enhanced their granulocyte formation, and this enhancement was observed even after a prolonged maintenance without growth factors. The spherical bodies were composed of a uniform mixture of endothelial cells and fibroblasts within the body, and cell-cell contacts between lymphocytes and fibroblasts were notable in the periphery. With prolonged culture, the spherical bodies showed a definite change in their structure by sorting two cell types and the blood cell production gradually decreased. These results suggested that a three-dimensional structure was required for the maintenance, growth, and differentiation of blood cell progenitors in the long-term spleen culture.

Gamma interferon responses of spleen cells in mice were examined during postchemotherapy relapse of intraperitoneally induced latent tuberculous infection. The mycobacterial extract RUTI, which prevented the relapse, significantly enhanced the immune responses to secreted and structural recombinant mycobacterial antigens, suggesting that RUTI-mediated protection was mediated by activated T cells.

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.

For the past 2 years, the mouse spleen has been the organ most thoroughly investigated to elucidate the role of cytokines and chemokines in influencing the structure of secondary lymphoid organs during ontogeny and in the adult state. Mice with deficiencies of cytokine genes and animals injected with soluble cytokine receptors before birth have provided the first evidence that the normal development of splenic compartments depends on the action of diffusible (or membrane-bound) mediators. At present, there are two types of molecules, cytokines of the TNF family and several chemokines, which have been shown to influence the compartmentalisation of the spleen in mice.

En el presente estudio se analizo la respuesta eritropoyética a la anemia inducida por flebotomía crónica y los cambios en la distribución del hierro celular y sistémico del organismo. Ratones hembra de la cepa CF1 (n=32), se dividieron en dos lotes: control y experimental, siguiendo un diseño experimental pareado según su peso. La distribución del hierro en bazo e hígado durante la flebotomía crónica fue evaluada mediante estudios morfológicos y la actividad eritropoyética mediante estudios hematológicos. Las diferencias estadísticas se determinaron mediante Test Student. El nivel de significancia se fijó en p

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.

A dual origin, by both immigration of blood monocytes and local proliferation has been reported for macrophages, which are heterogeneous in several aspects. Until now few studies have focussed on renewal of macrophages. Therefore, in this study macrophage renewal by local proliferation has been studied in the spleen. Spleen tissue of mice which had received BrdU intravenously was investigated immunohistochemically to detect BrdU incorporated by macrophages. BrdU incorporation by splenic macrophages was studied under steady state conditions as well as during repopulation after experimental macrophage depletion. Under steady state conditions, BrdU incorporation was found in 3.0 ± 0.5% of the white pulp macrophages and in approximately 1% of the metallophilic macrophages only. Comparable results were found during repopulation after experimental macrophage depletion. The data suggest that renewal of macrophages by local proliferation is restricted to certain subsets in the spleen and that the macrophage subsets of the spleen are renewed by different mechanisms, or belong to different lineages of macrophage differentiation.

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.

Computer assisted nonlinear regression analysis has been used to examine the time course of the in vivo accumulation of labelled lymphocytes in lymphoid tissues after their intravenous transfer to syngeneic recipients. We examined the time course of rat thoracic duct lymphocytes (TDL) reported by Smith and Ford (1), and the localization of murine CD4 enriched lymphocytes in our own experiments. Quantitative estimates for the clearance rate of these lymphocyte populations from the blood and the residency time of the lymphocytes in spleen and Peyer’s patches were obtained using a simple model of lymphocyte migration kinetics and commercially available software.

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