Number Of Spleen Cells Research Articles

IL-15 Can Signal via IL-15Rα, JNK, and NF-κB To Drive RANTES Production by Myeloid Cells

IL-15 plays many important roles within the immune system. IL-15 signals in lymphocytes via trans presentation, where accessory cells such as macrophages and dendritic cells present IL-15 bound to IL-15Rα in trans to NK cells and CD8(+) memory T cells expressing IL-15/IL-2Rβ and common γ chain (γ(c)). Previously, we showed that the prophylactic delivery of IL-15 to Rag2(-/-)γ(c)(-/-) mice (mature T, B, and NK cell negative) afforded protection against a lethal HSV-2 challenge and metastasis of B16/F10 melanoma cells. In this study, we demonstrated that in vivo delivery of an adenoviral construct optimized for the secretion of human IL-15 to Rag2(-/-)γ(c)(-/-) mice resulted in significant increases in spleen size and cell number, leading us to hypothesize that IL-15 signals differently in myeloid immune cells compared with lymphocytes, for which IL-15/IL-2Rβ and γ(c) expression are essential. Furthermore, treatment with IL-15 induced RANTES production by Rag2(-/-)γ(c)(-/-) bone marrow cells, but the presence of γ(c) did not increase bone marrow cell sensitivity to IL-15. This IL-15-mediated RANTES production by Rag2(-/-)γ(c)(-/-) bone marrow cells occurred independently of the IL-15/IL-2Rβ and Jak/STAT pathways and instead required IL-15Rα signaling as well as activation of JNK and NF-κB. Importantly, we also showed that the trans presentation of IL-15 by IL-15Rα boosts IL-15-mediated IFN-γ production by NK cells but reduces IL-15-mediated RANTES production by Rag2(-/-)γ(c)(-/-) myeloid bone marrow cells. Our data clearly show that IL-15 signaling in NK cells is different from that of myeloid immune cells. Additional insights into IL-15 biology may lead to novel therapies aimed at bolstering targeted immune responses against cancer and infectious disease.

Abstract 1351: Analysis of the in vivo tumor suppressive potential of PTPROt (truncated protein tyrosine phosphatase receptor-type O) in CLL using a transgenic mouse model

Abstract Protein tyrosine phosphatases (PTPs) and kinases (PTKs) and their corresponding substrates are integrated within elaborate signal transduction networks essential for regulating several important cellular functions including growth, differentiation, cell cycle, gene transcription, the immune response and survival. Defective operation of these networks leads to aberrant tyrosine phosphorylation that contributes to the development of many human diseases including cancer. Importantly, as observed with PTKs, deregulation of PTPs plays a key role in the pathogenesis of these diseases. Thus, PTPs and their substrates represent novel molecular targets for the development of agents in the treatment of these diseases. We have previously demonstrated that the gene encoding the hematopoietic-specific truncated isoform of protein tyrosine phosphatase receptor-type O (PTPROt), a phosphatase demonstrating growth suppressor characteristics, is suppressed in chronic lymphocytic leukemia (Clin Cancer Res. 2007 Jun 1;13(11):3174-81). We have further shown that expression of PTPROt is severely impeded by T-cell leukemia 1 (TCL1), an oncoprotein aberrantly expressed in CLL B-cells (Blood. 2011 Oct 14. [Epub ahead of print]). Additionally, PTPROt plays an important role in regulating B-cell receptor (BCR) signaling (J Cell Biochem. 2010 Jul 1;110(4):846-56; Blood. 2006 Nov 15;108(10):3428-33). To comprehend the physiological relevance of PTPROt we have now generated transgenic (Tg) mice expressing PTPROt in B-cells. These mice live their normal life span and exhibit normal B- and T-cell development. Lyn and SYK kinases, the established substrates of PTPROt, are hypo-phosphorylated in B-lymphocytes from the spleen of PTPROt Tg mice relative to those from NTg mice. Because expression of PTPROt is significantly lower in splenic B-lymphocytes from TCL1 Tg mouse model of CLL relative to splenic B-cells from non-transgenic (NTg) mice (Blood. 2011 Oct 14. [Epub ahead of print]), we crossed the TCL1 Tg and PTPROt Tg mice to determine whether expression of PTPROt will inhibit/delay TCL1-mediated leukemogenesis and utilize this model to elucidate the mechanism of PTPROt function in maintaining normal cell physiology. Preliminary gene expression analysis performed on splenic B-cells from NTg, TCL1 Tg and PTPROt/TCL1 double Tg mice demonstrated that the key positive regulators of cell cycle elevated in the TCL1 Tg mice (relative to the NTg mice) are downregulated in the PTPROt/TCL1 double Tg mice (relative to the TCL1 Tg mice). Further, the spleen size as well as the total number of spleen cells was significantly lower in the PTPROt/TCL1 double Tg group relative to the TCL1 Tg group. We are currently in the process of assessing the function of the altered genes in the TCL1 Tg mouse model as well as in human CLL. [Supported by grant CA101956] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1351. doi:1538-7445.AM2012-1351

Population dynamics of leukocytes in blood during early activation of the chicken immune system by E. coli

The immune system must have appropriate levels of nutrients to support changes in leukocyte numbers during immune response, but quantitative aspects are poorly characterized. Thus, examined the early time course of peripheral blood leukocytes of adult Hyline chickens injected IV with dead E. coli. After primary injection, blood was collected and total white blood cells (WBC), CD4+, CD8+), IgM+, IgG+), and macrophage/monocyte numbers were determined. WBCs decreased at 4hr, 18hr, 24hr, and 48hr post‐injection (PI) compared to baseline (P<0.0001 for first 3, P=0.04). CD4+ decreased compared to baseline at 4hr, 18hr, 24hr, and 48hr PI (P<0.0001 for first 3, P=0.01). CD8+ decreased compared to baseline at 4hr, 18hr, 24hr, and 48hr PI (P<0.0001 for first 3, P=0.0001). IgM+ decreased compared to baseline at 4hr, 18hr, 24hr, and 48hr PI (P<0.001 for first 3, P=0.004). IgG+ decreased at 4hr, 18hr, 24hr, and 48hr PI compared to baseline (P<0.0001 for first 3, P=0.0009). Macrophage/monocytes decreased at 4hr, 18hr, 24hr, and 48hr PI compared to baseline (P=0.0008, P=0.003, P=0.008, P=0.01). Maximal percent decrease in cell numbers at 4hr for WBC was 96%. Decrease of cellular components in blood occurred, observed an increase in cell number in spleen, thymus and bone marrow. Converted to a whole body basis, the 70% decrease is less than the changes observed in other tissues indicating a decrease in nutrient use due to activation.Grant Funding Source: USDA Regional Research project 1013

Early Life Interventions to Prevent Allergy in the Offspring: The Role of Maternal Immunization and Postnatal Mucosal Allergen Exposure

Background: Childhood allergy is influenced by maternal factors and allergen exposure in early life, but the factors that determine the development of allergy and tolerance are unknown. Therefore, we compared the effects of two early life interventions, i.e. maternal allergen immunization and postnatal intranasal allergen exposure, as well as a combination of both treatments on allergic responses in the offspring. Methods: Female mice were immunized with ovalbumin (OVA) or vehicle during pregnancy. After birth, half the offspring from each group were exposed intranasally to low doses of OVA or vehicle weekly for 5 weeks before intraperitoneal immunization with OVA. Results: Maternal immunization reduced OVA-specific IgE and IgG1, but increased IgG2a and T_H2 cytokine responses in the offspring after immunization. Postnatal intranasal OVA exposure similarly reduced both IgE and IgG1, but also spleen cell numbers and cytokine secretion. Following airway challenges of the offspring, IgE and airway inflammation were suppressed only by intranasal exposure, but not by maternal immunization, the effect of which also waned with age. Differential gene expression in the spleen of offspring supported that IgE suppression by the two interventions was caused by different mechanisms. Despite this, tolerance development after mucosal exposure was obtained in the offspring of immunized dams. Conclusions: The study suggests that prevention of allergy is possible if initiated in early life, and early mucosal allergen exposure may play a protective role independent of the maternal immune responses.

Interleukin‐15 suppresses hepatitis B virus replication via IFN‐β production in a C57BL/6 mouse model

Interleukin-15 (IL-15) is a pleiotropic cytokine known to modulate both innate and adaptive immunity. It is suggested that IL-15 may play an important role in the regulation of immune response to hepatitis B virus (HBV). We investigated whether IL-15 could modulate the immune response to HBV. A mouse model for HBV tolerance was established by hydrodynamical injection of pAAV/HBV1.2 plasmid into C57BL/6 mice. This HBV-carrier mouse was simultaneously hydrodynamically injected with either an IL-15-expression plasmid pLIVE-IL-15 or a mock control vector pLIVE-EGFP. The serum levels of HBsAg and HBeAg were measured by radioimmunoassay. Hydrodynamic injection of the plasmid pLIVE-IL-15 resulted in sustained high level of IL-15 in mouse serum, along with the markedly decreased serum HBsAg and HBeAg titres and liver HBV DNA levels. IL-15 also induced anti-HBV activity in T cell- and B cell-deficient Rag1(-/-) mice. Interestingly, despite an increase in NK cell numbers in both spleen and liver of IL-15 treated mice, the anti-HBV effect of IL-15 was neither dependent on presence of NK cells nor on production of IFN-γ. Furthermore, IL-15 could exert anti-HBV function independent of the common IL-2γ(c) R. Lastly, we found that IFN-β expression in the liver and serum was significantly up-regulated by liver expression of IL-15, and blockade of IFN-β function abrogated the anti-HBV activity of IL-15. Liver over-expression of IL-15 may suppress HBV replication in an IFN-β-dependent manner.

Antitumor and Immunostimulating Activities of Elfvingia applanata Hot Water Extract on Sarcoma 180 Tumor-bearing ICR Mice

Elfvingia applanata, a medicinal mushroom belonging to Basidiomycota, has been used in the effort to cure cancers of the esophagus and stomach, and is also known to have inhibitory effects on hepatitis B virus infection. The hot water soluble fraction (as Fr. HW) was extracted from fruiting bodies of the mushroom. In vitro cytotoxicity tests showed that hot water extract was not cytotoxic against cancer cell lines such as Sarcoma 180, HT-29, HepG2, and TR at concentrations of 10~2,000 μg/mL. Intraperitoneal injection with Fr. HW resulted in a life prolongation effect of 45.2% in mice previously inoculated with Sarcoma 180. Treatment of Fr. HW resulted in a 2.53-fold increase in the numbers of murine spleen cells at a concentration of 50 μg/mL, compared with control. Incubation of murine spleen cells with Fr. HW at a concentration of 500 μg/mL resulted in improved immune-potwntiating activity of B lymphocytes through an 8.3-folds increase in alkaline phosphatase activity, compared with control. Fr. HW generated 12.5 μM of nitric oxide (NO) when cultured with RAW 264.7, a mouse macrophage cell line, at the concentration of 50 μg/mL, while lipopolysaccharide, a positive control, produced 15.2 μM of NO. Therefore, the results suggested that antitumor activities of Fr. HW from E. applanata might, in part, be due to host mediated immunostimulating activity.

PSGL-1 Regulates the Migration and Proliferation of CD8+ T Cells under Homeostatic Conditions

P-selectin glycoprotein ligand-1 (PSGL-1), a heavily glycosylated sialomucin expressed on most leukocytes, has dual function as a selectin ligand for leukocyte rolling on vascular selectins expressed in inflammation and as a facilitator of resting T cell homing into lymphoid organs. In this article, we document disturbances in T cell homeostasis present in PSGL-1(null) mice. Naive CD4(+) and CD8(+) T cell frequencies were profoundly reduced in blood, whereas T cell numbers in lymph nodes and spleen were at or near normal levels. Although PSGL-1(null) T cells were less efficient at entering lymph nodes, they also remained in lymph nodes longer than PSGL-1(+/+) T cells, suggesting that PSGL-1 supports T cell egress. In addition, PSGL-1(null) CD8(+) T cell proliferation was observed under steady-state conditions and PSGL-1(null) CD8(+) T cells were found to be hyperresponsive to homeostatic cytokines IL-2, IL-4, and IL-15. Despite these disturbances in T cell homeostasis, PSGL-1(null) mice exhibited a normal acute response (day 8) to lymphocytic choriomeningitis virus infection but generated an increased frequency of memory T cells (day 40). Our observations demonstrate a novel pleiotropic influence of PSGL-1 deficiency on several aspects of T cell homeostasis that would not have been anticipated based on the mild phenotype of PSGL-1(null) mice. These potentially offsetting effects presumably account for the near-normal cellularity seen in lymph nodes of PSGL-1(null) mice.

Megalocytivirus 감염 해산 어류에서 나타나는 임상증상의 정량적 변화 분석

In quantitative studies of clinical signs, rock bream of adults and juveniles infected with Megalocytivirus IVS-1 isolated from rock bream (Oplegnathus fasciatus) in Korea showed average and of spleen index respectively. In challenge experiments, Megalocytivirus IVS-1 induced 100% cumulative mortality in both adult and juvenile rock bream. However we found 60% cumulative mortality in juvenile red sea bream (Pagrus major) even after 30 days of injection, which contradicted with the results of other laboratories. Interestingly, IVS-1 infected red sea bream of the same juvenile size with rock bream showed lower spleen index compared to that of rock bream. In real-time PCR, there was continuous increasing of the numbers of viral copies ( copies/mg) in the spleen of juvenile rock bream infected, which were different from those in adult rock bream showing plateau level after reaching to the peak level. Moreover, enlarged cell numbers in the infected spleen were also increased continuously in the juvenile but not in adult of rock bream, even decreased after reaching to peak level. Consequently, significant differences in clinical signs: cumulative mortality. spleen index and viral copy number were found between rock bream and red sea bream, but not between adult and juvenile rock bream. Certainly quantitative expression of clinical sign as in this study may be a way to compare the progression of megalocitiviral disease more accurately in different species or physiological conditions.

Role of acute ethanol exposure and TLR4 in early events of sepsis in a mouse model

Sepsis is a major cause of death worldwide. The associated risks and mortality are known to significantly increase on exposure to alcohol (chronic or acute). The underlying mechanisms of the association of acute ethanol ingestion and poor prognosis of sepsis are largely unknown. The study described here was designed to determine in detail the role of ethanol and TLR4 in the pathogenesis of the sepsis syndrome. The effects of acute ethanol exposure and TLR4 on bacterial clearance, spleen cell numbers, peritoneal macrophage numbers, and cytokine production were evaluated using wild-type and TLR4 hyporesponsive mice treated with ethanol and then challenged with a nonpathogenic strain of Escherichia coli. Ethanol-treated mice exhibited a decreased clearance of bacteria and produced lesser amounts of most pro-inflammatory cytokines in both strains of mice at 2 h after challenge. Neither ethanol treatment nor a hyporesponsive TLR4 had significant effects on the cell numbers in the peritoneal cavity and spleen 2 h postinfection. The suppressive effect of acute ethanol exposure on cytokine and chemokine production was more pronounced in the wild-type mice, but the untreated hyporesponsive mice produced less of most cytokines than untreated wild-type mice. The major conclusion of this study is that acute ethanol exposure suppresses pro-inflammatory cytokine production and that a hyporesponsive TLR4 (in C3H/HeJ mice) decreases pro-inflammatory cytokine levels, but the cytokines and other mediators induced through other receptors are sufficient to ultimately clear the infection but not enough to induce lethal septic shock. In addition, results reported here demonstrate previously unknown effects of acute ethanol exposure on leukemia inhibitory factor and eotaxin, and provide the first evidence that interleukin (IL)-9 is induced through TLR4 in vivo.

Overexpression of short CYLD leads to autoimmunity and production of autoantibodies (47.20)

Abstract CYLD is a tumour suppressor gene which plays an important role in NF-kB signaling. To analyze the function of CYLD in vivo we used the CYLDex7/8 mouse strain, which is characterized by loss of the full-length transcript and overexpression of a naturally occurring short splice variant of the cyld gene (sCYLD). This overexpression resulted in splenomegaly and lymphadenopathy. Further, the B cell numbers in spleen and lymph nodes were increased at the expense of T cells. CYLDex7/8 mice showed a strong reduction of CD4 single positive (SP) and CD8 SP T cells in the thymus and periphery. In in vitro and in vivo studies we could show that CD4+ T cells display a hyperactive phenotype accompanied with an increased production of inflammatory cytokines. We could also demonstrate a defect in negative selection of CYLDex7/8 thymocytes by using the HY-TCR transgenic system. mTECs in the thymus, which are important for the negative selection of autoreactive T cells, were drastically reduced in number. Since abnormal T cell responses are often associated with chronic inflammations, we examined if CYLDex7/8 mice develop immunological abnormalities. Importantly, we could observe that CYLDex7/8 mice crossed onto TCR transgenic background developed severe colonic inflammation accompanied with high production of nuclear autoantibodies.

Plasmacytoid dendritic cell depletion augments CD8 T cell IFN-γ associated atherogenic responses in a mouse model of atherosclerosis. (147.18)

Abstract Plasmacytoid dendritic cells (pDC) have been shown to play a pivotal immunogenic role in viral immune responses by releasing high IFN-alpha levels upon TLR9 activation. However, evidence is culminating that pDC also play a major role in inducing immune tolerance in chronic low grade inflammation. In this study we set out to address effects of pDC depletion on atherosclerosis development. Carotid artery lesions were induced in LDLr-/- mice by perivascular collar placement. To study effects of pDC depletion on atherogenesis, a pDC depleting antibody, 120G8, was administered daily for 4 weeks. GL113 mAb was used as isotype control. pDC depletion resulted in exacerbated atherosclerosis, characterized by an increased lesion CD3+ T cell infiltration. FACS analysis showed an increase in CD3+ T cell numbers in spleen and lymph nodes with a concurrent shift towards CD8+ T cells. This was paralleled by strongly elevated plasma IFN-γ levels. Moreover, immature but not mature pDC from spleen of atherosclerotic mice expressed increased levels of IDO and PD-L1 compared to pDC from non-atherosclerotic mice. Finally, we found that pDC isolated from human UAP/SAP patients displayed reduced IFNalpha expression, while intralesional expression of IFNalpha was unaltered at later stages of disease progression. In conclusion, our data demonstrate that immature pDC act atheroprotective by dampening CD8+ T cell responses and keeping Th2/Th1 responses in check.

Recipient CD73-generated adenosine plays a preventive role in graft-versus-host disease by regulating allogeneic T cell activation (169.16)

Abstract Graft-versus-host disease (GvHD) is a systemic disease in which donor T cells attack the allogeneic recipient. Here we investigated the role of CD73 in GvHD induced by transplantation of BALB/c bone marrow and spleen cells into lethally irradiated C57BL/6 recipients. CD73 is an enzyme that generates extracellular adenosine, a nucleoside with anti-inflammatory and immunosuppressive activity mediated by adenosine receptors. Survival curves showed more severe GvHD in CD73-/- → CD73-/- transplants than in CD73+/+ → CD73+/+ transplants and serum IFNγ and IL-6 were higher in CD73-/- recipient mice. CD4+ and CD8+ T cells from CD73-/- donors proliferated more in vivo than those from CD73+/+ donors as assessed by CFSE dye dilution and cell numbers in spleens. However, irradiation-induced activation of dendritic cells as assessed by CD86 up regulation was similar in CD73+/+ and CD73-/- mice. In vivo anti-C57BL/6 CTL activity was higher in CD73-/- transplant recipients than in CD73+/+ recipients. Additional CD73-/- → CD73+/+ and CD73+/+ → CD73-/- transplants revealed that more severe GvHD is caused primarily by a lack of CD73 in recipients. The adenosine receptor antagonist caffeine exacerbated GvHD in CD73+/+ recipients, consistent with the idea that the protective effect of CD73 depends upon its adenosine-generating activity. In conclusion, recipient CD73 plays a primary preventive role in GvHD by regulating allogeneic donor T cell activation in an adenosine-dependent manner.

Low dose γ-radiation can mitigate some deleterious effects of carcinogen injection in A/J mice. (165.42)

Abstract Low dose radiation (LDR) activates the immune response and may lead to cancer suppression. We investigated the acute effect of LDR and the carcinogen benzo(a)pyrene (BaP) on lung and spleen cell number and phenotype as well as spleen cell cytokine secretion in an A/J mouse model. Mice were given a single gamma radiation dose (10, 100, 1000 mGy) one day prior to intraperitoneal injection with BaP or Tricaprylin vehicle. Lung and spleen tissues were harvested at days (d) 2, 7, and 9 post-irradiation to determine viable cell counts and phenotype. Splenocytes were cultured for 48 hours +/- Concanavalin A (5 μg/ml) and cytokine secretion was measured by Luminex. BaP treatment decreased lung cell (d2) and splenocyte (d7) number while lung neutrophila was increased on d7 and 9. BaP also decreased activated (CD69+) CD4+ and CD8+ cells in both organs by d7. The reduction in activated CD8+ was attenuated in mice treated with 10 mGy radiation. BaP also decreased lung B cell and dendritic cell number. Treatment with 1000 mGy decreased B cells in both the lung and spleen. BaP treatment decreased Th2 cytokine production while increasing IL-1β and IL-17. 10 mGy treatment increased IL-2, IL-4, IL-12p70, VEGF, and IL-17 while IL-6 and IL-10 production was decreased by higher radiation doses on d2 post-radiation. These data suggest that treatment with BaP and LDR significantly affect the immune system with 10 mGy, but not 1000 mGy, treatment often mitigating the deleterious effect of BaP.

DDE and diet-induced obesity alter thymocyte and splenocyte immune cell populations and renal expression of osteopontin and MCP-1 in a murine model (44.31)

Abstract DDE, a metabolic breakdown product of the pesticide DDT, accumulates in adipose tissue and acts as an anti-androgen. Since androgens can protect against autoimmuity, DDE may impact autoimmmune developement. We predicted greater effects of DDE in mice with more adipose tissue. Male C57BL/6J mice were fed either a high-fat (60 kcal% fat) or control low-fat(10 kcal% fat) diet from weaning. At 8 weeks, mice were treated with DDE (200 mg/kg body wt)or vehicle by oral gavage. Mice were sacrificed at 10 weeks or 6 months. Body weights were at least 50% greater in high-fat diet mice. Total spleen and thymus cell numbers were not altered by diet or DDE. Fewer mature thymus T cells (CD3+) were found in the DDE high fat group compared with the DDE low fat group (25.22 +/- 2.76 % vs. 47.55 +/- 3.69%, p<0.05) at 10 weeks. High fat diet, independent of DDE treatment resulted in more CD8+ single-positive thymocytes in high fat controls compared with low fat controls (2.04 +/- 0.9 % vs. 1.13 +/- 0.13%, p<0.05) at 10 weeks and more CD4+ splenocytes in high fat compared with low fat controls (25.63 +/- 2.67% vs. 16.30 +/- 0.79%, p<0.05) at 6 months. DDE caused a 10-15% increase in lymphocytic infiltrates in kidney glomeruli regardless of diet. High fat diet and DDE treatment increased renal expression of MCP-1 while DDE treatment increased OPN expression. In conclusion, diet and obesity influence some effects of DDE on the immune system. Study of additional immune parameters is warranted.

잎새버섯(Grifola frondosa)의 자실체에서 추출한 조다당류의 면역증강 및 항암효과

잎새버섯의 자실체로부터 메탄올, 중성염용액 및 열수를 이용하여 조다당류를 추출하고 ICR mouse에 주사하여 항암 및 면역증강 효과를 조사하였다. 잎새버섯 조다당류의 Sarcoma 180 및 RAW 264.7에 대해 세포독성을 조사한 결과 10~2000 <TEX>${\mu}g/ml$</TEX>의 조다당류 농도에서 각각의 세포는 독성을 나타내지 않았고, HT-29 및 NIH3T3 세포의 경우에는 중성염용액으로 추출한 조다당류 10~1000 <TEX>${\mu}g/ml$</TEX>의 농도에서는 독성을 보이지 않았으나 2000 <TEX>${\mu}g/ml$</TEX>의 농도에서는 약한 독성을 나타내었다. Sarcoma 180으로 접종된 ICR mouse에 잎새버섯의 자실체에서 추출한 각각의 조다당류를 투여한 실험군은 조다당류를 투여하지 않은 대조군에 비해 평균수명이 각각 25.0~52.9% 연장되었다. 열수로 추출한 조다당류를 200 <TEX>${\mu}g/ml$</TEX>의 농도를 처리한 비장세포 수는 대조군에 비하여 1.3배 증가하였으며, 200 <TEX>${\mu}g/ml$</TEX>의 농도로 중성염추출 조다당류를 투여한 실험군 생쥐 B 임파구의 alkaline phosphatase 활성은 대조군에 비해 약 1.5배 증가하였다. 대식세포 RAW 264.7에 의해 발생된 nitric oxide(NO)의 농도는 대조군이 4.3 <TEX>${\mu}M$</TEX>인 것에 비해 중성염 추출 조다당류를 50~500 <TEX>${\mu}g/ml$</TEX>의 농도로 처리한 실험군의 대식세포는 10~14 <TEX>${\mu}M$</TEX>의 nitric oxide를 발생시켰다. 메탄올, 중성염용액 및 열수로 추출한 조다당류로 처리한 비장세포는 모든 농도에서 대조군에 비해 TNF-<TEX>${\alpha}$</TEX>, IL-<TEX>$1{\beta}$</TEX>, IL-2, IL-6 등의 사이토카인 분비량이 1.4배 이상 높았다. 메탄올 추출 조다당류를 50 mg/kg body weight의 농도로 투여한 실험군의 총 복강세포의 수와 백혈구의 수는 대조군에 비하여 각각 3.0배, 2.0배 증가하였다. 따라서 잎새버섯의 자실체에서 추출한 조다당류는 생쥐의 면역을 증강시키고 Sarcoma 180에 대한 항암효과가 있는 것으로 사료된다. 80% methanol and 0.9% neutral saline soluble and hot water substances (hereinafter referred to Fr. NaCl, Fr. HW and Fr. MeOH, respectively) were extracted from fruiting bodies of Grifola frondosa. In vitro cytotoxicity tests, crude polysaccharides were not cytotoxic against cancer cell lines such as Sarcoma 180 and RAW 264.7 at the concentration of 10~2000 <TEX>${\mu}g/ml$</TEX>, but crude polysaccharides from Fr. NaCl was slightly toxic to HT-29 and NIH3T3 at the concentration of 2000 <TEX>${\mu}g/ml$</TEX>. Intraperitoneal injection with crude polysaccharides exhibited life prolongation effect of 25.0~52.9% in mice previously inoculated with Sarcoma 180. Fr. HW increased the numbers of spleen cells by 1.3 fold at the concentration of 200 <TEX>${\mu}g/ml$</TEX> compared with control. Fr. NaCl improved the immuno-stimulating activity of B lymphocyte by increasing the alkaline phosphatase activity by 1.5 fold compared with control at the concentration of 200 <TEX>${\mu}g/ml$</TEX>. 10~14 <TEX>${\mu}M$</TEX> of nitric acid were generated when Fr. NaCl was added to RAW 264.7 at the concentration of 50~500 <TEX>${\mu}g/ml$</TEX>, while the control group produced 4.3 <TEX>${\mu}M$</TEX> of nitric oxide. The Fr. NaCl, Fr. HW and Fr. MeOH increased the production of TNF-<TEX>${\alpha}$</TEX>, IL-<TEX>$1{\beta}$</TEX>, Il-2 and IL-6 by more than 1.4 times compared with the control group. The Fr. of MeOH increased the numbers of peritoneal exudate cells and circulating leukocytes by 3.0 and 2.0 folds compared with the control at the concentration of 50 <TEX>${\mu}g/ml$</TEX>, respectively. Therefore, the crude polysaccharides extracted from fruiting bodies of Grifola frondosa could improve antitumor activity of mice.

Induction of protective immunity against Eimeria tenella infection using antigen-loaded dendritic cells (DC) and DC-derived exosomes

Current methods for sustainable control of avian coccidiosis, whether by prophylactic medication or parasite vaccination, are suboptimal. In this study, we describe an alternative immunization strategy against Eimeria tenella infection using parasite antigen (Ag)-loaded dendritic cells (DCs), or their derived exosomes, in the absence of free Ag. CD45 + intestinal DCs were isolated from E. tenella-infected chickens and loaded ex vivo with an extract of sporozoites as parasite Ag. Extracellular vesicles purified from the Ag-pulsed DCs expressed surface proteins associated with DC-derived exosomes, including major histocompatibility complex proteins (MHC I and MHC II), CD80, flotillin, and heat shock protein (HSP70). Following intramuscular immunization of chickens with Ag-pulsed DCs or Ag-pulsed DC-derived exosomes, Ag-containing cells were observed diffusely localized in the lymphoid tissue and concentrated in germinal centers of caecal tonsils, and restricted to germinal centers (GC) in the spleen. Chickens immunized with pulsed DCs or exosomes exhibited (a) higher numbers of caecal tonsil and spleen cells expressing IgG and/or IgA antibodies that were reactive with E. tenella Ag, (b) greater numbers of IL-2-, IL-16-, and IFN-γ-producing cells, and (c) higher E. tenella Ag-driven cell proliferation, compared with chickens immunized with Ag in the absence of DCs or exosomes. Chickens immunized with Ag-pulsed DCs or Ag-pulsed DC-derived exosomes and subsequently given a live E. tenella challenge infection at 10 d post-immunization displayed (a) increased body weight gains, (b) decreased feed conversion ratios, (c) reduced fecal oocyst shedding, (d) diminished intestinal lesions, and (e) lower mortality, compared with animals given Ag alone. This is the first demonstration of Ag-specific protective immunity against avian coccidiosis using parasite Ag-loaded DCs or DC-derived exosomes.

Transgenic expression of bovine neonatal Fc receptor in mice boosts immune response and improves hybridoma production efficiency without any sign of autoimmunity

The overexpression of the bovine neonatal Fc receptor (bFcRn) in transgenic (Tg) mice boosts humoral immune response with increased numbers of antigen-specific spleen cells and a potent humoral immune response against weakly immunogenic targets. One of the interesting questions surrounding this enhanced immune response is whether these Tg mice generate higher number of antigen-specific hybridomas. To address this question, we immunized these Tg mice and wild type (wt) controls with trinitrophenylated proteins, generated hybridomas and analyzed their numbers and specificities. We observed that Tg mice generated a 3–5 fold increase in antigen-specific IgG titers and had significantly larger spleens containing higher number of antigen-specific B cells and plasma cells, analyzed by ELISA and ELISPOT assays. Fusion of the isolated splenocytes with standard mouse myeloma cells (SP2/0-Ag14) resulted in a 2–4 fold elevation of hybridization frequency for the hapten, or carrier-specific IgG positive microcultures, in Tg mice compared to controls. In addition, as augmented immune reactivity leads to autoimmunity in some genetically modified mouse strains, we analyzed autoreactive antibody levels in serum samples derived from elderly bFcRn Tg mice by a protein chip assay. In contrast to the sample from the MRL/lpr mouse suffering from autoimmunity, we did not detect autoantibodies in bFcRn Tg mice or the wt controls. Based on these and our earlier data, we propose that Tg mice that overexpress bFcRn offer major advantages in monoclonal Ab production.

Role of dihydrotestosterone in post-stroke peripheral immunosuppression after cerebral ischemia

Stroke is a sexually dimorphic disease with male gender considered a disadvantage in terms of risk and disease outcome. In intact males, stroke induces peripheral immunosuppression, characterized by decreased splenocyte numbers and proliferation and altered percentages of viable T, B, and CD11b+ cells. To investigate whether the potent androgen and known immunomodulator, dihydrotestosterone (DHT), exacerbates post-stroke immunosuppression in castrated male mice after focal stroke, we evaluated the effect of middle cerebral artery occlusion (MCAO) on peripheral and central nervous system (CNS) immune responses in castrated mice with or without controlled levels of DHT. MCAO reduced spleen cell numbers in both groups, but altered T cell and B cell percentages in remaining splenocytes and concomitantly increased the percentage of CD11b+ blood cells solely in DHT-replaced animals at 24h. Furthermore, DHT-replacement reduced splenocyte proliferation which was accompanied by an increased percentage of immunosuppressive regulatory T cells relative to castrates 96h post-MCAO. In brain, the percentages of immune cell populations in the ischemic hemisphere relative to the non-ischemic hemisphere were similar between castrated and DHT-replaced mice after MCAO. These data suggest DHT modulates peripheral immunosuppression after MCAO but with relatively little effect on early immune response of the recovering CNS.

Low-dose Photon and Simulated Solar Particle Event Proton Effects on Foxp3+ T Regulatory Cells and other Leukocytes

Radiation is a major factor in the spaceflight environment that has carcinogenic potential. Astronauts on missions are continuously exposed to low-dose/low-dose-rate (LDR) radiation and may receive relatively high doses during a solar particle event (SPE) that consists primarily of protons. However, there are very few reports in which LDR photons were combined with protons. In this study, C57BL/6 mice were exposed to 1.7 Gy simulated SPE (sSPE) protons over 36 h, both with and without pre-exposure to 0.01 Gray (Gy) LDR g-rays at 0.018 cGy/h. Apoptosis in skin samples was determined by immunohistochemistry immediately post-irradiation (day 0). Spleen mass relative to body mass, white blood cells (WBC), major leukocyte populations, lymphocyte subsets (T, Th, Tc, B, NK), and CD4(+)CD25(+)Foxp3+ T regulatory (Treg) cells were analyzed on days 4 and 21. Apoptosis in skin samples was evident in all irradiated groups; the LDR+sSPE mice had the greatest expression of activated caspase-3. On day 4 post-irradiation, the sSPE and LDR+sSPE groups had significantly lower WBC counts in blood and spleen compared to non-irradiated controls (p < 0.05 vs. 0 Gy). CD4(+)CD25(+)Foxp3(+) Treg cell numbers in spleen were decreased at day 4, but proportions were increased in the sSPE and LDR+sSPE groups (p < 0.05 vs. 0 Gy). By day 21, lymphocyte counts were still low in blood from the LDR+sSPE mice, especially due to reductions in B, NK, and CD8(+) T cytotoxic cells. The data demonstrate, for the first time, that pre-exposure to LDR photons did not protect against the adverse effects of radiation mimicking a large solar storm. The increased proportion of immunosuppressive CD4+CD25(+) Foxp3(+) Treg and persistent reduction in circulating lymphocytes may adversely impact immune defenses that include removal of sub-lethally damaged cells with carcinogenic potential, at least for a period of time post-irradiation.

Post-Transplant Cyclophosphamide Treatment Ameliorates Experimental Gvhd While Permitting Lymphopenic Expansion of Non-Host Reactive Donor T Cells.

AbstractAbstract 3751Following myeloablative as well as reduced intensity conditioning regimens, recipients of allogeneic T cell replete (TCR) HLA matched sibling)or unrelated hematopoietic stem cell transplants (HSCT) experience significant acute as well as chronic graft vs. host disease (GVHD). Recent studies have reported that post-transplant cyclophosphamide (PTC) can function as an effective single agent for prophylaxis of acute and chronic GVHD (Biol. Blood Marrow Transpl. 16:482, 2010; Blood. 116:3224, 2010). To investigate the fate of non-host alloreactive donor cells, which are critical to providing immune reconstitution post-transplant, T cell replete HSCT were performed between MHC-matched allogeneic donor–recipient pairs. C3H.SW (H2b) mice were lethally conditioned 4 hrs prior to receipt of B6-CD45.1 (H2b) T cells and bone marrow. Recipients developed severe GVHD as assessed by weight loss (Fig. 1A) and clinical analyses during the first 4–5 wks post-transplant. Recipients had inverted CD4/CD8 ratios as well as an activated phenotype (CD44hiCD62Llo), severe B and T cell lymphopenia, and virtually undetectable thymic tissue. A single dose of post-transplant cyclophosphamide at day 3 (66mg/kg/body weight) blocked onset of weight loss and clinical signs of GVHD as well as lethality in C3H.SW recipients, consistent with the reduction of alloreactive anti-recipient T cells. In contrast to non-cyclophosphamide treated animals, treated recipients expressed typical CD4/CD8 ratios, significant numbers of spleen and lymph node cells (Fig. 1B) and readily detectable thymi 7–8 weeks post-HSCT. Notably, the majority of T as well as B cells in the periphery of these recipients exhibited a non-activated phenotype and were derived from donor bone marrow. Together with the observation that approximately 50% of T cells exhibited a CD44hiCD62Llo phenotype characteristic of TN, the findings are consistent with de novo derived lymphopoiesis in the thymus and marrow of post-transplant cyclophosphamide treated recipients. To assess the impact of D.3 cyclophosphamide treatment on a non-host reactive T cell population, B6 OT-I CD8 T cells (Vα2/Vβ5 TcR) were added (1-2×106) to the donor inoculum. In non-PTC treated recipients, low numbers of OT-I T cells (below input levels) were detected by 3–4 weeks post-HSCT. In contrast, in PTC-treated recipients, significant numbers of OT-I CD8 T cells were present in the recipient spleen and lymph nodes (Fig. 1C), consistent with the finding of CFSE labeled OT-I cells exhibiting division following cyclophosphamide treatment. Notably, the overall numbers of OT-I CD8 T cells in cyclophosphamide treated recipients was greater than the input levels, indicating that these cells a) survived post-cyclophosphamide treatment and b) underwent lymphopenic expansion. Consistent with these findings, CFSE studies at 72 hrs post-HSCT illustrated OT-I T cells exhibited low proliferation vs. a population of presumed B6 anti-C3H.SW alloreactive T cells. We therefore conclude that non-host reactive donor T cells undergo sufficient repair following cyclophosphamide induced alkylation to enable greater survival in contrast to rapidly dividing anti-host (GVHD) reactive populations. Vaccination studies in cyclophosphamide treated and non-treated HSCT recipients are being performed using heat shock fusion protein-transfected tumor cells to investigate the capacity to elicit anti-tumor (i.e., GVL) responses in the presence and absence of GVHD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.