Effect of Ionizing Radiation on the Expression of p16, CyclinDI and CDK4 in Mouse Thymocytes and Splenocytes

Objective To investigate the effect of ionizing radiation on the expression of p16, CyclinDl, and CDK4 in mouse thymocytes and splenocytes. Methods Fluorescent staining and flow cytometry analysis were employed for the measurement of protein expression. Results In time course experiments, it was found that the expression of p16 protein was significantly increased at 8, 24, and 48 h for thymocytes (P<0.05, P<0.01, and P<0.05, respectively) and at 24 h for splenocytes (P<0.05) after whole body irradiation (WBI) with 2.0 Gy X-rays. However, the expression of CDK4 protein was significantly decreased from 8 h to 24 h for thymocytes (P<0.05,P<0.01) and from 8 h to 72 h for splenocytes (P<0.05-P<0.01). In dose effect experiments, it was found that the expression of p16 protein in thymocytes and splenocytes was significantly increased at 24 h after WBI with 1.0, 2.0, and 4.0 Gy (P<0.05-P<0.01), whereas the expression of CDK4 protein was significantly decreased with 2.0Gy for thymocytes (P<0.05) and 0.5-6.0 Gy for splenocytes (P<0.05-P<0.01). Results also showed that the expression of CyclinDl protein decreased markedly in both thymocytes and splenocytes after exposure. Conclusion The results indicate that the expression of p 16 protein in thymocytes and splenocytes can be induced by ionizing radiation, and the p16-CyclinD1/CDK4 pathway may play an important role for G1 arrest of thymocytes induced by X-rays.

Immunomodulating property of Ocimum sanctum by regulating the IL-2 production and its mRNA expression using rat's splenocytes

Objective:To investigate the regulatory effect of aqueous extract of leaves of Ocimum sanctum on IL-2 cytokine production in vivo and in vitro,and the effect of leaves extract on general blood picture including T& B lymphocytes.Methods:For in vivo studies albino Wistar rats were treated with aqueous crude leaves extract of Ocimum sanctum for 20 consecutive days.Spleen cells were harvested and assayed for IL-2 production by using sandwich enzyme-linked immunosorbent assay(ELISA) and mRNA expression methods.For in vitro study aqueous Ocimum sanctum leaves extract= in different concentrations(25-500μ/mL) was added into culture plates containing ConA stimulated splenocytes.To study the overall effect on blood picture,density gradient purified lymphocytes analysis and conventional methodology for total and differential leukocyte count and hemoglobin level were also done.Results:It indicated that the rats treated with Ocimum sanctum leaves extract had significantly enhanced(P【0.001) ability of spleen cells to secrete IL-2.Investigation in vitro also showed regulation of IL-2 production.Blood study exhibited leucocytosis and augmentation of T& B lymphocytes by 25%approximately.4-5%increase in Hemoglobin value was also noticed.Conclusion:Aqueous Ocimum sanctum leaves extract may have stimulatory effect on T & B lymphocytes particularly on Th 1 subset of lymphocytes as shown by enhancement in IL-2 production.

A murine model of mixed bone marrow transplant:importance of the splenocytes to balance HVG and GVH reactions

Objective: A murine model of mixing syngeneic and haploidentical major histocompatibility complex (MHC) matched bone marrow cells transplant was used to evaluate the effect of splenocytes in graft-versus-host disease (GVHD) and host-versus-graft reaction (HVGR). Methods: BALB/C recipient mice were lethally conditioned with 8.5 Gy and injected with different grafts which consisted of syngeneic bone marrow cells plus splenocytes (SPLCs) and haploidentical MHC matched bone marrow cells (BMCs)plus different doses of splenocytes. Recipient mice were detected for the percentage of haploidentical MHC matched mouse origin cells in the peripheral blood cells and checked daily for the appearance of GVHD symptoms. Histopathological examination of multiple organs from moribund mice was used to evaluate the grades of GVHD. Results: Recipient mice infused with 10 × 106 haploidentical MHC matched SPLCs and 5×106 syngeneic splenocytes showed a higher level and more stable chimerism with GVHD Ⅱ degree histopathological alterations. Histopathological results of GVHD in other group's hosts were not obvious, and the levels of chimerism were unstable. All of the mice survived over 150 d. Conclusion:The proportion and dose of syngeneic and haploidentical MHC splenocytes are of importance for inducing stable engraftment on the basis of nonlethal GVHD and to balance GVHD and HVGR.

PROLIFERATION OF ANTI－CD_3 McAb AND IL－2 INDUCED SPLENOCYTES AND ANTITUMOR EFFECT OF THEIR CULTURE SUPERNATANTS

The proliferation of splenocytes from health adults was induced by anti-CD3 McAb and IL-2.The proliferative potential of the splenocytes and antitumor activity of their culture supernatants of splenocytes were studied.The results showed that anti-CD3 McAb not only enhanced the proliferation of the splenocytes directly,but also enhanceil that of induced by IL-2.Their enhancing effect was more significant when the incubation time in vitro was prolonged.The culture supernatants of anti-CD3 and IL-2 induced splenocytes also had the antitumor activity and enhancing capability to the antitumor activity of LAK tells.The results suggested that LAK cells could secret lymphokine,and this effect would be synergically promoted when anti-CD3 and IL-2 were simultaneously used.

RESPONSES OF HUMAN FETAL SPLENOCYTES AND THYMOCYTES TO INTERLEUKIN-2: LAK ACTIVITY AND PROLIFERATION

Using cytotoxicity and thymidine uptake assays, we investigated the effects of human recombinant in-terleukin-2 (rIL-2) on the induction of lympholine-activated killer (LAK) activity and cellular proliferation in splenocytes and thymocytes from human fetuses (18-22 weeks). We observed that fetal splenocytes and thymocytes incubated with low doses of rIL-2 (10-100 U ml) developed broad antitumor activity (LAK activity) although the kinetics and magnitudes of the responses were different. It indicated the LAK precursors are present in fetal spleen and thymus. Further, rIL-2 induced a strong proliferative response in splenocytes, but not in thymocytes. On the basis of the findings, we conclude that the responses of fetal splenocytes and thymocytes to IL-2 are different.

Differential Effects of Ursolic Acid and Oleanolic Acid on Mitochondrial ATP Generation in H9c2 Cardiomyocytes and Lipopolysaccharide-Induced Cell Proliferation in Mouse Splenocytes: Yang versus Yin

In the present study, two cell-based systems for assessing Yang and Yin activities were for the first time used to investigate the effect of ursolic acid (UA) and oleanolic acid (OA). The results indicated that while UA was only active in the Yang assay, OA produced activity in the Yin assay. The Yang/Yin activity of UA/OA may be attributed to their distinct molecular structures, which confer their differential ability to interact with mitochondrial membrane or cellular membrane lipids, with resultant membrane fluidization and potentiation of biological responses.

Immunopotentiating Activity of Dendrobium Species in Mouse Splenocytes

This study aimed to explore a pharmacological activity marker for quality assurance of Dendrobium species. The immunopotentiating activity in aqueous extracts prepared from four Dendrobium species, including D. officinalis, was assessed by an in vitro assay of concanavalin A (Con A)-stimulated proliferation of mouse splenocytes. Four samples of commercially available Dendrobii Caulis were also analyzed for comparison. The results indicated that the aqueous extract of D. officinalis produced immunopotentiating action, as evidenced by the increase in Con A-stimulated proliferation of mouse splenocytes, with the extent of stimulation being more prominent than those of other tested Dendrobium species and Dendrobii Caulis samples. In conclusion, an in vitro immunopotentiation assay may be used for assessing the pharmacological activity of Dendrobium species. The finding that D. officinalis produced a more potent immunopotentiating action is consistent with its 'yin-nourishing' action in Chinese medicine, which is more effective than other Dendrobium species in clinical use.

Remarkably Decreased CD11b Positive Splenocytes in Aquaporin 3-Null Mice

Aquaporins（AQPs） are molecular water channels that play important physiological roles in fluid trans-porting organs. The expression and function of AQPs in the immune system are largely unknown. CD 11 （a-d）/CD18 integrins are adhesion molecules expressed on leukocytes, which play a critical role in leukocyte adhesion, migration and host defense. In the present study, we discovered the expression of aquaporin-3（AQP3） on spleen CD1 lb positive cells, and the content of CDllb positive splenocytes in aquaporin 3-null mice is significantly decreased. Further analysis suggested remarkably decreased monocyte/macrophage subpopulation and significantly decreased granulocyte subpopulation. It is the first report suggesting an important role of AQP in the development and maturation of imrnunocytes.

Pharmacological Investigation on the Qi-Invigorating Action of Schisandrin B: Effects on Mitochondrial ATP Generation in Multiple Tissues and Innate/Adaptive Immunity in Mice

Schisandrae Fructus, containing schisandrin B (Sch B) as its main active component, is recognized in traditional Chinese medicine (TCM) for its Qi-invigorating properties in the five visceral organs. Our laboratory has shown that the Qi-invigorating action of Chinese tonifying herbs is linked to increased mitochondrial ATP generation and an enhancement in mitochondrial glutathione redox status. To explore whether Sch B can exert Qi-invigorating actions across various tissues, we investigated the effects of Sch B treatment on mitochondrial ATP generation and glutathione redox status in multiple mouse tissues ex vivo. In line with TCM theory, which posits that Zheng Qi generation relies on the Qi function of the visceral organs, we also examined Sch B’s impact on natural killer cell activity and antigen-induced splenocyte proliferation, both serving as indirect measures of Zheng Qi. Our findings revealed that Sch B treatment consistently enhanced mitochondrial ATP generation and improved mitochondrial glutathione redox status in mouse tissues. This boost in mitochondrial function was associated with stimulated innate and adaptive immune responses, marked by increased natural killer cell activity and antigen-induced T/B cell proliferation, potentially through the increased generation of Zheng Qi.

Differential Effects of Yin- and Yang-Chinese Tonifying Herbs on Innate and Adaptive Immunity

The present study investigated the effect of treatment with methanolic extracts of Yin- and Yang-Chinese tonifying herbs on concanavalin A (Con A)/lipopolysaccharide (LPS)-stimulated splenocyte proliferation (adaptive immunity) and natural killer (NK) cell activity (innate immunity) in an ex vivo mouse model. The results indicated that while treatment with most Yin herbal extracts potentiated the Con A/LPS-stimulated splenocyte proliferation, only Yang (but not Yin) herbal extracts stimulated NK cell activity. The differential effects of Yin- and Yang-Chinese tonifying herbs on innate and adaptive immunity are consistent with the Chinese medicine theory which depicts the Yin and Yang functional components of Zheng Qi (vital energy), with the Yang component being responsible for the first line of defense against invading microorganisms (i.e., innate immunity) and the Yin oner serving as a follow-up defensive response (adaptive immunity).

Immunopotentiality of Ayurvedic polyherbal formulations “Saribadi” and “Anantamul Salsa” with augmentation of IgM production and lymphocytes proliferation:A preliminary study

Objective:To assess the immunopotentiality of Ayurvedic polyherbal preparations,"Saribadi" and "Anantamul Salsa".Methods: Freshly prepared BALB/c mice splenocytes were cultured with "Saribadi" or"Anantamul Salsa" treatment [doses of 0.25%, 0.50%, 0.75%, 1.00%, 1.50%, 2.00%,3.00% and 4.00%(v/v)] at 37C for 5 days. The immunoglobulin M(IgM) production and lymphocytes proliferation were determined by ELISA and MTT methods, respectively.Endotoxin contamination was assessed by treating the preparations with polymyxin B.Results: The doses of "Saribadi" [0.25%, 0.50%, 0.75% and 1.00%(v/v)] significantly increased IgM productions(0.966, 0.728, 0.695 and 0.615 mg/m L vs. control 0.265 mg/m L)and lymphocytes proliferation [absorbance 0.311, 0.394, 0.372 and 0.334 optical density(OD) vs. control 0.162 OD]. Similarly, the doses of "Anantamul Salsa" [0.50%, 0.75%,1.00% and 1.50%(v/v)] promoted IgM productions(0.933, 0.919, 0.917 and 0.892 mg/m L vs. control 0.502 mg/m L) and the doses of "Anantamul Salsa" [0.50%, 0.75%, 1.00%,1.50%, 2.00%, and 3.00%(v/v)] stimulated lymphocytes proliferation(absorbance 0.395,0.326, 0.440, 0.398, 0.452 and 0.355 OD vs. control 0.199 OD). The activity of "Saribadi"and "Anantamul Salsa" was not retarded by the treatment of preparations with polymyxin B.Conclusions: Immunomodulatory activity of "Saribadi" and "Anantamul Salsa" was unveiled for the first time. "Saribadi" and "Anantamul Salsa" possess immunostimulating potential acting through the induction of lymphocyte proliferation and IgM production.These preparations may be useful in strengthening immune responses. However, further cellular and in vivo studies are required.

Immunotoxicity assessment of cadinene sesquiterpenes from Eupatorium adenophorum in mice

Sesquiterpenes in Eupatorium adenophorum are abundant in leaves and have great development potential as biopesticides. The toxicity of sesquiterpenes in immune cells and their corresponding immune functions are not fully understood. We evaluated the immunotoxicity of two cadinene sesquiterpenes 2-deoxo-2-（acetyloxy）-9-oxoageraphorone（DAOA） and 9-oxo-10,11-dehydro-agerophorone（ODA） by using histopathology and toxicology methods in vitro and in vivo in lymphocytes and natural killer cells in Kunming mice. The mice were given single doses of 75, 150 and 300 mg kg^-1 body weight（BW） of DAOA/ODA every day for a week. S erious damage to the thymus and spleen was found in tissue images with clear lysis reduction numbers and a loosened arrangement of splenocytes and thymocytes to the mice treated with 150–300 mg kg^-1 DAOA/ODA. Mice cytology was also affected with significant cellular alterations, increased splenocytes apoptosis rates（P〈0.01）, proliferation reduction（P〈0.05） and natural killer cells activities reduction（P〈0.05） when given 150–300 mg kg^-1 DAOA/ODA, the severities of which were dose-dependent. Howev er, a 75 mg kg^-1 dose of DAOA/ODA showed no change in tissue or cytology after the 7 day treatment, and therefore was considered to be within acceptable safety parameters. Taken together, cadinene sesquiterpenes, as a type of toxic botanical component, have low environmental risks in small doses and should be further studied for their use as biopesticides.

Immune stimulatory activity of BRP-4,an acidic polysaccharide from an edible plant,Basella rubra L.

Objective:To evaluated the immunomodulatory effect of BRP-4,an acidic polysaccharide from Basella rubra(B.rubra) L on the macrophage activity.Methods:Phagocytic activity was determined by the ingestion of Latex Beads-Rabbit IgC-FITC using the fluorescent microscopy and flow cytometry analysis and nitric oxide production was measured using Griess reaction assay.Results:An enhanced production of NO was observed at 10 and 100 μg/mL of BRP-4.The phagocytic activity of macrophage was enhanced in BRP-4 treated RAW264.7 cells.BRP-4combined with concanavalin A(Con A) provided obvious promotion and strengthening of the proliferation of the splenocytes.Conclusions:BRP-4,polysaccharide isolated from B.rubra,is suggested to activate macrophage function and stimulate splenocyte proliferation.The strong immunomodulatory activity of BRP-4 confirmed its good potential as an immunotherapeutic adjuvant.

Formation of microchimerism in rat small bowel transplantation by spienocyte infusion

AIM： To investigate the effect of donor splenocyte infusion combined with cyclosporine A （CsA） on rejection of rat small bowel transplantation （SBT）. METHODS： Male Sprague-Dawley （SD） rats and female Wistar rats weighing 230-270 g were used as donors and recipients respectively in the study. Heterotopic small bowel transplantation was performed. The rats were divided into three groups： group one receiving allotransplantation （SD→Wistar）, group two receiving allotransplantation （SD→Wistar） ＋ donor splenocyte infusion, group three receiving allotransplantation （SD →Wistar） ＋ donor splenocyte infusion ＋ CsA followed by CsA 10 mg/kg per day after transplantation, in which recipient Wistar rats were injected with 2 ×10^8 SD splenocytes 28 d before transplantation, and treated with CsA after transplantation. Finally, the specific DNA fragment of donor Y chromosome was detected in recipient peripheral blood and skin by PCR. The survival time after small bowel transplantation was observed. Gross and histopathological examinations were performed. RESULTS： The survival time after small bowel transplantation was 7.1 ± 1.2 d in group 1, 18.4 ± 3.6 d in group 2 and 31.5± 3.1 d in group 3. The survival time was significant longer （P 〈 0.01） in group 3 than in groups 1 and 2. The gross and histopathological examination showed that the rejection degree in group 3 was lower than that in groups 1 and 2.CONCLUSION： Donor splenocyte infusion combined with CsA decreases remarkably the rejection and prolongs the survival time after rat small bowel transplantation.

Nano chloroquine delivery against Plasmodium berghei NK65 induced programmed cell death in spleen

Objective:To compare the protective effects of chitosan-trypolyphosphate(CS-TPP) nanoparticle conjugated chloroquine(CQ) with effect of CQ alone on the reversal of splenic damages and induction of apoptosis.Methods:Different researches have been carried out to explore the potential role of chitosan based drug delivery system against parasitic diseases.After successive Plasmodium berghei NK65 parasiste infection by intraperitoneal injection in Swiss mice and subsequent parasite development,the ROS generation,anti-apoptotic and pro apoptotic protein levels in spleen were measured.To analyze caspases,flow cytometry study was performed with annexin 桋-FITC and with PI staining.Results:The results revealed that ROS mediated caspase 3 and 9 activation and the induction of apoptosis occurred during the parasitic infection.However,CS-TPP conjugated CQ was relatively better in reversing the splenic damage compared with similar effects of CQ alone.Conclusions:This study indicates that Plasmodium berghei NK65 induces apoptosis in the spleen.The study further shows that CS-TPP nanoparticles conjugation with CQ have positive influence on the recovery of damaged host's system towards maintenance of normal homeostasis,and this is shown to be selective to CS-TPP conjugated CQ treated animals only.

Pharmacological Investigation of “Meridian Tropism” in Three “Shen” Chinese Herbs

“Meridian tropism” refers to the organ-specific biological action(s) produced by a Chinese herb following its oral administration, which is analogous to the concept of “bioavailability” in Western medicine. In this study, we compared the in vitro and ex vivo pharmacological actions of three herbs [namely, Dangshen (DS, Codonopsis Radix), Ranshen (RS, Ginseng Radix) and Xiyangshen (XYS, Panacis Qinquifolii Ra-dix)] to validate their meridian tropism. We compared the in vitro and ex vivo pharmacological actions [i.e. the ability to increase splenocyte proliferation and adenosine triphosphate-generation capacity (ATP-GC)] of the ethanolic extracts of DS, RS and XYS to validate their meridian tropism. Results showed that DS, RS and XYS (at 30 - 300 μg/mL) can both stimulate the proliferation of primary mouse splenocytes in vitro and increase adenosine triphosphate-generation capacity (ATP-GC) in cultured Caco 2 colon epithelial cells in vitro. Interestingly, oral administration of DS and RS (but not XYS, at 3 and 6 g/kg/day × 3 consecutive days) was found to stimulate the proliferation of splenocytes ex vivo at 24 h post-treatment in mice. Similarly, DS and RS (but not XYS) increased the ATP-GC of mitochondrial fractions isolated from a small segment of mouse intestine at 48 h post-treatment. This observation is consistent with the meridian tropism of the pharmacological action of “Shen”, i.e., the accessibility of DS and RS (but not XYS) to the “Spleen” meridian. The comparison between the results obtained from in vitro and in vivo/ex vivo bioassays may offer a potential method for assessing meridian tropism in Chinese herbs.

Comparison of the characteristics of macrophages derived from murine spleen, peritoneal cavity, and bone marrow

Macrophages have a diverse set of functions based upon their activation states. The activation states, including resting（M0） and polarizing（M1 and M2） states, of macrophages derived from the mouse bone marrow, spleen, and peritoneal cavity（BMs, SPMs, and PCMs, respectively） were compared. We evaluated the macrophage yield per mouse and compared the surface markers major histocompatibility complex（MHC） Ⅱ and CD86 by flow cytometry. The relative mRNA levels of tumor necrosis factor-α（TNF-α）, interleukin（IL）-1β, mannose receptor（MR）, and Ym1 in the M0, M1, and M2 states were also compared using real-time polymerase chain reaction（PCR） analysis. Bone marrow yielded the most macrophages with the best homogeneity, but they were polarized toward the M2 phenotype. All three types of macrophages had the capacity to polarize into the M1 and M2 states, but SPMs had a stronger capacity to polarize into M1. The three types of macrophages showed no differences in their capacity to polarize into the M2 state. Therefore, the three types of macrophages have distinct characteristics regardless of their resting or polarizing states. Although bone marrow can get large amounts of homogeneous macrophages, the macrophages cannot replace tissue-derived macrophages.

Combination of donor splenocyte transfusion with blockade of γc signal synergizes to inhibit alloreactive T-cell proliferation and induces apoptosis

Background The common y chain （γc） plays a critical role in regulating proliferation, differentiation, and apoptosis of peripheral T-cells. It was previously confirmed that blocking the yc signal can successfully induce transplant tolerance in a murine model. Here we investigated the potential mechanism. Methods Splenocytes from C57BL/6 mice were transfused into T-cell deficient Balb/c nude mice that were reconstituted with syngeneic wild-type T-cells labeled with 5-carboxyfluorescein diacetate succinimidyl ester （CFSE）. After 24 hours, recipients received i.p. injection of mixture of anti-γc mAbs, or with isotype control IgG2a. The labeled T-cells were harvested from recipient spleens after 12 and 48 hours. T-cell proliferation and apoptosis were detected by flow cytometry. Results T-cell proliferation was markedly inhibited and apoptotic T cells could be detected at 12 hours after the mAbs injection. Proliferation was inhibited at 48 hours, but the proportion of apoptotic T-cells was not more than at 12 hours. In the control group, however, T-cells actively proliferated and no significant apoptosis was detected at either time point. Conclusions The results suggested that blockade of γc signals can synergize with donor splenocyte transfusion and lead to inhibition of antigen-specific T-cell proliferation and induction of apoptotic T-cell death. This protocol may develop a novel approach to induce donor-specific tolerance.

Activation of Signal Transducer and Activator of Transcription 5 (STAT5) in Splenocyte Proliferation of Asthma Mice Induced by Ovalbumin

To investigate the role of signal transducer and transcriptional activator 5(STAT5)activated in ovalbumin (OVA)-induced splenocyte proliferation of asthma mice,an asthma mouse model was set up by intraperitoneal injection and aspiration of OVA with nebulizer.The proliferation of splenocytes isolated from the asthma mice was detected by [~3H]thymidine incorporation.The phosphorytation of STAT5 was examined by Western blotting and STAT5-DNA binding was measured by electrophoretic mobility shift assay(EMSA).OVA could pronouncedly induce the splenocyte proliferation of asthma mice in a dose-dependent manner compared with control groups.Phosphorylation of STAT5 and STAT5-DNA binding were observed in splenocytes from asthma mice induced by OVA at 1 h and 3 h.These results indicated that STAT5 signal pathway played an important role in lymphocyte proliferation of asthma mice induced by OVA.Cellular & Molecular Immunology. 2004;1(6):471-474.

Isolation and purification of BV Ⅰ-2H from bee venom and analysis of its biological action

The medical use of bee venom for rheumatoid arthritis ( RA ) has a very long tradition. In this study, isolation and purification of polypeptides from bee venom were carried out on sephadex chromatography, heparin sepharose CL6B chromatography and HPLC. Several fractions were extracted, and their effects on activation of splenocyte and THP-1 cell were studied. The inhibitory fraction was selected for further studies. Finally, BV I-2H that the HPLC elution profiles was a single peak was isolated by C8 column. ESI-MS detection results showed that BY I-2H was a fraction of bee venom, and the molecular weight of the major component was 644.8. BY I -2H could inhibit ConA-induced splenocyte proliferation, IL-1 production and interfere with splenocyte cycle in mice. Moreover, BV I-2H could inhibit PMA-induced TNFa production in THP-1 cells, which was due to its inhibitory effects on TNFa mRNA expression and protein phosphorylation of IκBα. Our studies indicated that BY I -2H was one of the anti-inflammatory