Number Of Peritoneal Exudate Cells Research Articles

Characterization of immunomodulatory responses induced by manuka honey.

Manuka honey (MH) is known for its wound-healing, anti-microbial, anti-oxidant and anti-tumor properties. However, there is conflicting evidence regarding the role of MH in inflammatory responses, with some studies highlighting its pro-inflammatory capacity and others showing that it has a predominantly anti-inflammatory activity. The current study is aimed at characterizing the immunomodulatory capacity of MH using both in vitro and in vivo approaches, focusing on the underlying mechanisms. Treatment of RAW 264.7 macrophages with 1% MH (w/v) resulted in a significant increase in the gene expression (~26-fold) and secretion (~27-fold) of tumor necrosis factor-alpha (TNF-α). Similarly, an increase was observed in the gene expression of other inflammatory cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS), as well as the chemokines; (C-X-C motif) ligand 2 (CXCL2) and (C-C) motif ligand 2 (CCL2). Using an in vivo model, intraperitoneal (i.p.) administration of MH in C57BL/6 mice elicited a peritoneal response characterized by a significant expansion in the number of peritoneal exudate cells (PECs), which was mainly due to a 35-fold increase in the recruitment of neutrophils. Importantly, this response was evident in toll-like receptor 4 (TLR4)-defective C3H/HeJ mice, indicating that the observed stimulatory effect occurs independently of TLR4 and unlikely to be mediated by any lipopolysaccharide (LPS) contaminant. MH administration also led to changes in the phenotypic expression and functional maturation of peritoneal macrophages, as evidenced by a shift towards the CD11blo F4/80lo phenotype and an increase in the expression of major histocompatibility complex (MHC) class II proteins. In contrast, the MH-initiated peritoneal response was largely abrogated in mice deficient in myeloid differentiation primary response 88 (MyD88) protein, a critical adaptor of most TLR signaling pathways. Thus, the current findings help to characterize the immunostimulatory properties of MH and their dependence on TLR signaling, and highlight the potential utility of MH as an immunomodulatory agent in a variety of disorders.

Choline metabolism promotes M2 macrophage polarization in intestinal infection with helminth Heligmosomoides polygyrus

Abstract Choline is an essential nutrient functioning as a precursor for membrane phospholipid. In previous studies, LPS polarization augmented both the expression of the choline transporter CTL1 and choline uptake, while specific inhibition of choline transport led to increased TNFα and IL-6. However, whether choline metabolism regulates M2 macrophage polarization or Th2 cytokine inflammation in vivo is unclear. In vitro, CTL1 and phospholipid synthesis was induced in IL-4 polarized M2 macrophages. To determine the function of choline metabolism in M2 polarization in vivo, mice were infected with Heligmosomoides polygyrus, intraperitoneally injected with choline kinase α inhibitor, RSM-932A or vehicle, and sacrificed at day 17 post-infection. RSM-932A treatment impaired weight gain and peritoneal exudate cell numbers in both naïve and infected mice. Within the peritoneal cavity of infected mice, macrophages and B-1 lymphocytes were depleted by RSM-932A treatment, while monocytes and neutrophils were increased. Flow cytometric and intestinal immunofluorescence staining revealed that RSM-932A treatment prevented M2 macrophage polarization in H.polygyrus-infected mice with a significant reduction in expression of PD-L2 and CD206, and conversely increased expression of CD86 and PD-L1. Additionally, RELMα protein in the serum and peritoneal fluid was downregulated by RSM-932A treatment. The impaired M2 polarization was associated with some loss in optimal immunity to H.polygyrus with increased parasite egg burden but no differences in intestinal worm count. This study indicates that choline metabolism is required for M2 macrophage polarization and an optimal immune response against intestinal helminth infection.

Leukocyte Factor XII Mediates Inflammation and Its Deficiency Promotes Wound Healing

Abstract 616 Background:Older investigations suggest that Factor XII (FXII) influences the inflammatory response. FXII deficient patients have reduced leukocyte migration into skin windows. In addition to bradykinin generation, FXII regulates the expression of monocyte FcγII receptor and stimulates monocytes and macrophages to release interleukin (IL)-1 and IL-6. In vitro, purified FXIIa corrects neutrophil aggregation and degranulation defects in FXII-deficient plasma. Our laboratory demonstrated that FXII signals through uPAR, β1 integrin and the EGFR to stimulate ERK1/2 and Akt phosphorylation (Blood 115:5111, 2010). The downstream consequences of this pathway are FXII-induced cell proliferation, growth and angiogenesis. Since FXII modulates wound angiogenesis, we examined the role for FXII in the inflammatory response and wound repair. Investigations:Exon 3 to 8–deleted FXII knockout mice (FXII−/−) were wounded by creating full thickness (5 mm) punch biopsies on their backs. The total surface area of inflammatory cells recruited to the injury site per 20X high power field (HPF) was determined and analyzed by Image J (NIH). We observed that on Day 1, FXII−/− mice exhibit significantly decreased recruitment of CD11 b–labeled inflammatory cells to injury sites compared with wild type mice (WT) [p=0.0136]. Similar results are observed when uPAR KO mice were compared to WT [p=0.0001], suggesting that leukocyte migration in part is mediated through this receptor. Next we determined the nature of cells in the wound sites. FXII−/− mice have reduced wound neutrophils (Gr-1 staining) and macrophages (F4-80 staining). On the thioglycolate (TG)-induced peritoneal inflammation assay, the number of peritoneal exudate cells (PECs) was measured in WT and FXII−/− mice 1 and 7 days following instillation. FXII−/− mice exhibit significantly decreased number of PEC’s on days 1 and 7. Giemsa-Wright stain of peritoneal lavage fluid with manual differential counts or flow cytometry shows that there is a disproportionate decrease in neutrophil recruitment in peritoneal fluid of FXII−/− mice. Also, FXII−/− macrophages have reduced adherence to plastic. Identical assays performed on uPAR and bradykinin B2 receptor (B2R) KO mice did not reveal the same inflammatory defects; both uPAR and B2R KO mice have mild decreases in PEC cell numbers following TG instillation, but overall their inflammatory response remained intact. Investigations next determined if the leukocyte migration defect seen in FXII−/− mice is related to plasma FXII or an intrinsic leukocyte defect. On adoptive bone marrow (BM) transplantation experiments, WT bone marrow transplanted to FXII−/− mice corrects the TG-induced PEC migration defect after both 1 and 7 days from instillation. Alternatively, transplantation of FXII−/− bone marrow into WT mice produced a leukocyte migration defect. In order to further discriminate the contribution of FXII in leukocyte migration, we produced plasma FXII deficiency in WT mice using FXII siRNA. WT mice treated with FXII siRNA reduced plasma FXII activity to <10% at 24 h (T1/2= 6 h). These mice did not have a defect in PEC recruitment. In vivo infusion of purified FXII did not correct the leukocyte peritoneal migration defect in FXII−/− mice. Next we harvested BM from WT mice and isolated leukocytes to prepare bone marrow cell-derived mRNA. The mRNA was reverse-transcribed to cDNA and it demonstrates XII cDNA that shares sequence homology to hepatic XII from exons 2 through 14. Immunofluorescence staining of BM-derived leukocytes demonstrates XII-positive leukocytes with nuclear morphology resembling monocytes and neutrophils (Fig. 1). Finally although leukocyte FXII promotes their migration into wounds, FXII deficiency in leukocytes paradoxically is associated with improved rates of wound healing after punch biopsy. Conclusions:These data indicate that there is a unique pool of FXII in leukocytes distinct from plasma (hepatic) FXII. Leukocyte FXII, not plasma FXII, is responsible for leukocyte function in wounds and inflammation sites. FXII−/− mice have attenuated wound injury and, paradoxically, improved wound healing rates. Modulation of leukocyte FXII is a target for promotion of wound healing. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Factor XII Promotes Leukocyte Inflammation and Its Deficiency Results in Faster Wound Healing

Abstract 368 Background:Factor XII (FXII) participates in inflammation. FXII deficient patients have reduced leukocyte migration into skin windows. Recently, we have determined that FXII signals through uPAR, β1 integrin and the EGFR to stimulate ERK1/2 and Akt phosphorylation (Blood 115:5111, 2010). The downstream consequences of this pathway are FXII-induced cell proliferation and post-natal angiogenesis. Present studies examined if this pathway also influences leukocyte response to injury. Methods:Exon 3- to 8-deleted FXII knockout mice (FXII KO), generously provided by Dr. Frank Castellino, have impaired inflammatory response to injury. FXII KO were wounded by creating full thickness (5 mm) punch biopsies on their backs. The total surface area of inflammatory cells recruited at the injury site/20 × high power field (HPF) was determined and analyzed by Image J (NIH). We observed that FXII KO on Day 1 exhibit significantly decreased recruitment of CD11b-labeled inflammatory cells to injury sites compared with wild type mice (WT) [mean ± SEM, FXII KO: 40220 ± 3732 vs. WT: 59740 ± 6318 total surface area of CD11b positive cells (pixels)/HPF, p=0.0136]. Similar results are observed when uPAR KO were compared to WT mice [39380 ± 5234 vs. 73310 ± 4688, p=0.0001], suggesting that leukocyte migration is mediated through uPAR. On Day 1 of thioglycolate (TG)-induced peritonitis, FXII KO have a mean of 12.62 × 105/mL peritoneal exudate cells (PEC) vs. 25.94 × 105/mL (p=0.0072) observed in WT. Additionally on Day 7, FXII KO have 10.63 × 105/mL PEC vs. WT 25.38 × 105/mL (p< 0.0001). Likewise on Day 7, uPAR KO have 16.34 × 105/mL peritoneal leukocytes recruited vs. WT 25.38 × 105/mL (p=0.0124). These combined studies indicate that FXII influences the degree of leukocyte inflammation and the inflammatory response is partially mediated through uPAR. Studies next determined if leukocyte recruitment promotes wound healing. On Days 3–5 the rate of wound closure of back punch biopsies is faster in FXII KO than WT (p < 0.0327 by one way ANOVA). Again, we observed that FXII KO on Days 2 and 5 exhibit significantly less CD11b-labeled inflammatory cells to the injury site compared to WT (Day 2: p=0.0004, Day 5: p= 0.0075). Leukocyte subpopulation analysis reveals decreased neutrophil migration (Gr-1 positive cells) into the wound area of FXII KO on Day 2 [FXII KO 5669 ± 1844 vs. WT 40490 ± 8564 total surface area of Gr-1 positive cells (pixels)/ HPF, p=0.0018] and Day 5 [FXII KO 6216 ± 3829 vs. WT 34890 ± 7629 total surface area of Gr-1 positive cells (pixels)/HPF, p=0.0176]. Macrophage recruitment into the wound area, as determined by F4-80 antigenicity, increases over time, but remains significantly reduced in FXII KO mice on Day 2 [FXII KO 3111 ± 1115 vs. WT 19140 ± 5767 total surface area of F4-80 positive cells (pixels)/HPF, p=0.0348] and Day 5 [FXII KO 9377 ± 4772 vs. WT 28340 ± 4411 total surface area of F4-80 positive cells (pixels)/HPF, p=0.0171]. These studies indicate that, in FXII deficiency, there is less neutrophil and macrophage-mediated inflammation and this observation correlates with faster wound healing. Finally studies determined if the reduced inflammation seen in FXII KO is the result of host factors or bone marrow-derived cells. Adoptive bone marrow transplant (BMT) experiments were performed where WT or FXII KO bone marrow was transplanted into FXII KO hosts. Eight weeks following the BMT, mice were subjected to TG-induced peritonitis. KO/KO recipients, have significantly decreased number of PEC on day 1 (17.3 × 105/mL ± 3.894 × 105/mL cells) and day 7 (18.6 × 107/mL ± 4.0 × 107/mL cells) when compared to WT/KO recipients [Day 1: 68.25 × 105/mL ± 13.83 × 105/mL cells (p=0.041), Day 7: 190.5 × 107/mL ± 51.80 × 107/mL cells]. These data indicate that FXII interacts with neutrophils and macrophages to promote the inflammatory response; its absence causes decreased inflammatory cell recruitment. Conclusions:Our data indicate that FXII deficiency disrupts the leukocyte response to injury by reducing inflammatory cell recruitment in two murine models. Paradoxically, reduced leukocyte infiltration into skin wounds promotes healing. These investigations indicate a novel role for FXII in inflammation and wound healing and indicate a unique potential target for inflammation therapeutics. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

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.

신령버섯(Agaricus brasiliensis) 자실체 추출 조다당류의 항암 및 면역증강 작용

신령버섯은 담자균문, 주름버섯목, 주름버섯과에 속하는 식용버섯으로 혈당, 혈압강하효과와 콜레스테롤 저하, 항종양, 암예방 효과가 있는 것으로 알려져 있다. 신령버섯의 자실체로부터 메탄올, 중성염용액 및 열수 등을 이용하여 조다당류를 추출하고 성분을 분석한 결과 <TEX>${\beta}$</TEX>-glucan은 21.54~32.31%, 단백질은 0.16~9.34%로 구성되어 있는 것이 밝혀졌고 신령버섯 추출 조다당류를 Sarcoma 180, HT-29, NIH3T3 및 RAW 264.7 등의 암세포 및 정상세포에 대한 독성을 조사한 결과 10~2000 <TEX>${\mu}g/ml$</TEX>의 조다당류 농도에서 각각의 세포는 세포독성을 나타내지 않았다. Sarcoma 180으로 접종된 ICR 생쥐에 신령버섯의 자실체에서 추출한 각각의 조다당류를 투여한 실험군은 대조군에 비해 수명이 각각 18.8~50.6% 연장되었다. 비장세포의 증식능과 B 임파구의 활성화에 미치는 alkaline phosphatase의 활성을 조사한 결과 비장 세포 증식능은 대조군에 대해 1.1~1.2배의 증식능을 보였고 B 임파구의 활성은 대조군에 대해 1.2~1.6배 증가하였다. 또한 버섯추출 조다당류를 투여한 실험군이 대조군에 비하여 대식세포에서 1.3~4.3배의 많은 nitric oxide를 발생시켰다. 비장세포에 여러 농도의 조다당류를 처리한 후 TNF-<TEX>${\alpha}$</TEX>, IL-<TEX>$1{\beta}$</TEX>, IL-2 및 IL-6 등의 사이토카인 분비량을 측정한 결과 생성된 사이토카인의 양은 대조군에 비해 2.2배 높게 나타났다. 중성염용액추출 조다당류를 50 mg/kg body weight의 농도로 투여한 실험군 생쥐의 총 복강 세포 수는 대조군에 비하여 4배 증가하였고, 열수추출 조다당류를 50 mg/kg body weight의 농도로 투여한 실험군 생쥐의 백혈구 수는 대조군에 비하여 각각 2.7배 증가하였다. 혈액생화학적 검사를 시행한 결과, 대조군과 유사한 경향을 나타냈다. 따라서 신령버섯의 자실체에서 추출한 조다당류는 생쥐의 면역을 증강시키는 것은 물론 Sarcoma 180에 대한 항암효과를 나타내는 것으로 밝혀졌다. Agaricus brasiliensis, one of edible mushroom belonging to Basidiomycota, has been used for curing gastric ulcer and stomach cancer of human beings and also known to have good inhibitory effects on sarcoma 180 and Ehrlich carcinoma of mice. Neutral saline soluble (0.9% NaCl), hot water soluble and methanol soluble substances (hereinafter referred to Fr. NaCl, Fr. HW and Fr. MeOH, respectively) were prepared from fruiting body of the mushroom. <TEX>${\beta}$</TEX>-glucan and total protein contents were identify from fractions of edible mushrooms extract. The <TEX>${\beta}$</TEX>-glucan and protein contents of all fractions of the mushrooms ranged from 21.54~32.31% and 0.16~9.34%, respectively. In vitro cytotoxicity tests, crude polysaccharides were not cytotoxic against cancer cell lines such as Sarcoma 180, HT-29, NIH3T3 and RAW 264.7 at the concentration of 10~2000 <TEX>${\mu}g/ml$</TEX>. Intraperitoneal injection with crude polysaccharides exhibited life prolongation effect of 18.8~50.6% in mice previously inoculated with Sarcoma 180. Fr. HW increased the numbers of spleen cell by 1.2 fold at the concentration of 200 <TEX>${\mu}g/ml$</TEX> compared with control. Fr. MeOH and Na improved the immuno-potentiating activity of B lymphocyte by increasing the alkaline phosphatase activity by 1.6 fold compared with control at the concentration of 50~500 <TEX>${\mu}g/ml$</TEX>. Fr. Na generated 15.9 <TEX>${\mu}M$</TEX> of nitric oxide (NO) when cultured with RAW 264.7 at the concentration of 200 <TEX>${\mu}g/ml$</TEX>, while lipopolysaccharide, a positive control, produced 3.7 <TEX>${\mu}M$</TEX>. The Fr. NaCl, Fr. HW and Fr. MeOH increased the secretion of TNF-<TEX>${\alpha}$</TEX>, IL-<TEX>$1{\beta}$</TEX>, Il-2 and IL-6 by 2.2 times compared with the control group. Fr. Na increased the numbers of peritoneal exudate cells by 4 folds at the concentration of 50mg/kg compared with control. Circulating leukocytes increased by 2.7 folds when Fr. HW from A. brasiliensis was inoculated at the concentration of 50 mg/kg body weight. The hematological and blood chemical analysis of the 3 fractions did not show any difference in blood compositions and enzyme activities compared with the control group (p<0.05). Therefore, the experimental results suggested that crude polysaccharides extracted from A. brasiliensis contain antitumor and immuno-potentiating activities against Sarcoma 180 in ICR mice.

노루궁뎅이(Hericium erinaceus) 자실체 추출 조다당류의 생쥐 Sarcoma 180에 대한 면역증강 및 항암 효과

노루궁뎅이는 담자균문, 민주름버섯목, 산호침버섯과에 속하는 식의약용버섯으로 예로부터 위궤양이나 위암의 치료에 효과가 있으며 생쥐의 Sarcoma 180과 Ehrlich Sarcoma에도 효과가 있다고 알려져 왔다. 노루궁뎅이의 자실체로부터 중성염용액, 열수 및 메탄올 등을 이용하여 조다당류를 추출하고 ICR mice에 주사하여 항암 및 면역증강 효과를 조사하였다. Sarcoma 180, HepG2, HT-29, NIH3T3 등의 세포에 대한 세포독성을 조사한 결과 <TEX>$10{\sim}2,000\;{\mu}g/ml$</TEX>의 조다당류 농도에서 각각의 암세포는 세포독성을 거의 나타내지 않았다. Sarcoma 180으로 접종된 ICR mouse에 자실체에서 추출한 각각의 조다당류를 투여한 실험군은 대조군에 비해 수명이 각각 29.1~54.1% 연장되었다. 노루궁뎅이 자실체를 중성염 용액으로 추출한 조다당류는 <TEX>$50\;{\mu}g/ml$</TEX>의 농도에서 비장세포의 수를 2.9배 증가시켰으며 또한 중성염 추출 조다당류를 <TEX>$200\;{\mu}g/ml$</TEX>의 농도로 투여한 실험군 생쥐의 B 임파구 alkaline phosphatase의 활성은 대조군에 비해 5.5배의 증가하였다. 중성염 용액 추출 조다당류를 50 mg/kg body weight의 농도로 투여한 실험군 생쥐의 총 복강 세포 수와 백혈구의 수는 대조군에 비하여 각각 4배와 2.3배로 증가하였다. 또한 면역 관련 장기인 간, 비장 및 흉선의 체중에 대한 무게 비율은 대조군에 비하여 유의성을 보이지 않았다. 따라서 노루궁뎅이의 자실체에서 추출한 조다당류는 생쥐의 면역을 증강시키고 생쥐의 Sarcoma 180에 대해 항암효과를 나타내었다. Hericium erinaceus, an edible and medicinal mushroom belonging to the Basidiomycota family, has been used for curing gastric ulcers and stomach cancers in human beings and is also known to have good inhibitory effects on sarcoma 180 and Ehrlich carcinoma in mice. Neutral saline soluble (0.9% NaCl), hot water soluble and methanol soluble substances (hereinafter referred to as Fr. NaCl, Fr. HW and Fr. MeOH, respectively) were extracted from the fruiting body of the mushroom. In in vitro cytotoxicity tests, crude polysaccharides were not cytotoxic against cell lines such as Sarcoma 180, HepG2, HT-29 and NIH3T3 at concentrations of <TEX>$10{\sim}2,000\;{\mu}g/ml$</TEX>. Intraperitoneal injection with crude polysaccharides exhibited a life prolongation effect of 29.1~54.1% in mice previously inoculated with Sarcoma 180. Fr. Na increased the numbers of spleen cells by 2.9 fold at a concentration of <TEX>$50\;{\mu}g/ml$</TEX> compared with the control. Fr. Na improved the immuno-potentiating activity of B lymphocytes by increasing alkaline phosphatase activity by 5.5 fold compared with the control at a concentration of <TEX>$200\;{\mu}g/ml$</TEX>. Fr. NaCl increased the numbers of peritoneal exudate cells and circulating leukocytes by 4 and 2.3 folds at a concentration of 50 mg/kg, respectively. Therefore, the crude polysaccharides extracted from the fruiting body of H. erinaceus could improve antitumor activities in mice.

Molecular events in the activation of B cells and macrophages by a non-microbial TLR4 agonist, G1-4A from Tinospora cordifolia

G1-4A, a polysaccharide from an Indian medicinal plant Tinospora cordifolia, was recently shown to protect mice against septic shock by modulating the proinflammatory cytokines. G1-4A also activated B cells polyclonally. The present report describes in detail the molecular events associated with G1-4A-induced immunomodulation in vitro and in vivo. G1-4A treatment led to an increase in the CD69 expression in lymphocytes. G1-4A-induced proliferation of B cells was completely inhibited by PI3K inhibitor Ly294002, mTOR inhibitor rapamycin and NF-κB inhibitor plumbagin. Akt, ERK and JNK were activated by G1-4A which finally resulted in the activation of IKK, degradation of IκB-α and translocation of NF-κB to the nucleus. Administration of G1-4A to mice led to splenomegaly and an increase in the numbers of T cells, B cells and macrophages. This increase in spleen cellularity was due to in vivo proliferation of lymphocytes and upregulation of anti-apoptotic genes. Anti-TLR4-MD2 complex antibody inhibited G1-4A-induced B cell proliferation and degradation of IκB-α suggesting that TLR-4 was a receptor for G1-4A on B cells. Activation of RAW 264.7 macrophages by G1-4A was found to be dependent on ERK and NF-κB-mediated signals. The phagocytosis index in peritoneal exudate cells (PEC) isolated from G1-4A treated mice was significantly higher as compared to that in PEC from control mice. G1-4A administration also increased the number of CD11b + cells in the PEC without an increase in the total number of PEC. Thus the present understanding of the molecular mechanism of action of G1-4A, a novel non-microbial TLR4 agonist, will pave the way for its application as an immunomodulator and adjuvant.

송이(Tricholoma matsutake)의 자실체에서 추출한 조다당류가 생쥐의 Sarcoma 180에 미치는 항암 및 면역증강 작용

송이는 담자균문, 주름버섯목, 송이과에 속하며 맛이 좋은 식용 버섯으로 예로부터 사람의 위궤양이나 위암의 치료효과는 물론 생쥐의 Sarcoma 180과 Ehrlich Sarcoma에도 효과가 있다고 알려져 있다. 본 연구에서는 송이의 자실체로부터 중성염용액, 열수 및 메탄올을 이용하여 조다당류를 추출하고 ICR mice에 주사하여 항암 및 면역증강 효과를 조사하였다. NIH3T3, Sarcoma 180, HepG2, HT-29 등의 암세포에 대한 독성을 조사한 결과 각각의 암세포는 2 mg/ml의 조다당류 농도에서는 세포독성을 나타내지 않았다. Sarcoma 180이 접종된 ICR mouse에 자실체에서 추출한 각각의 조다당류를 투여한 실험군은 대조군에 비해 평균수명이 각각 23.4<TEX>$\sim$</TEX>37.2% 연장되었다. 메탄올과 열수로 추출한 조다당류를 0.2<TEX>$\sim$</TEX>0.5 mg/ml의 농도로 투여한 실험군 생쥐의 B 임파구 alkaline phosphatase 활성은 대조군에 비해 각각 2.17<TEX>$\sim$</TEX>11.9배의 증가하였다. 중성염추출 조다당류를 50 mg/kg body weight의 농도로 투여한 생쥐의 총 복강 세포 수와 백혈구의 수는 대조군에 비하여 각각 6배와 1.5배 증가하였다. 따라서 송이의 자실체에서 추출한 조다당류는 생쥐의 Sarcoma 180에 대해 항암작용 및 면역증강 작용을 나타내는 것으로 확인되었다. Tricholoma matsutake, one of edible and medicinal mushroom belonging to Tricholomaceae of Agaricales, has been known to contain some curing effect on gastric cancer and ulcer, and inhibitory effect on sarcoma 180 and Ehrlich sarcoma. Neutral salt soluble (0.9% NaCl), hot water soluble and methanol soluble substances (hereinafter referred to Fr. NaCl, Fr. HW and Fr. MeOH, respectively) were extracted from fruiting body of the mushroom. In vitro cytotoxicity tests, crude polysaccharides were not cytotoxic against cancer cell lines such as Sarcoma 180, HepG2, HT29 and NIH3T3 at the concentration of 2.0 mg/ml. Intraperitoneal injection with crude polysaccharides showed life prolongation effect of 23.4<TEX>$\sim$</TEX>37.2% in mice previously inoculated with Sarcoma 180. Fr. MeOH and Fr. HW exhibited the immuno-potentiating activity of B lymphocyte by increasing the alkaline phosphatase activity by 2.2<TEX>$\sim$</TEX>11.9 folds compared with control at the concentration of 0.2<TEX>$\sim$</TEX>0.5 mg/ml. In case of Fr. NaCl, the numbers of peritoneal exudate cells and circulating leukocytes were increased by 6.0 and 1.5 folds at the concentration of 50 mg/kg, respectively. Therefore, it is concluded that crude polysaccharides extracted from fruiting body of Tricholoma matsutake showed antitumor and immuno-potentiating activity against Sarcoma 180 of mouse.

Dietary α-Linked Galacto-Oligosaccharide Suppresses Ovalbumin-Induced Allergic Peritonitis in BALB/c Mice

To determine whether alpha-linked galacto-oligosaccharide (alpha-GOS) prevents allergic peritonitis, BALB/c mice were fed a synthetic diet with and without alpha-GOS supplementation for 7 d, and were then subcutaneously immunized with ovalbumin on days 0 and 7. The mice were challenged by intraperitoneal injection with ovalbumin on day 14, followed by peritoneal lavage on day 15. The total number of peritoneal exudate cells was significantly lower in the mice fed the alpha-GOS diet than in those fed the control diet. Peritoneal lavage fluid from mice fed the alpha-GOS diet not only had less potency to attract peripheral blood leukocytes and peritoneal exudate cells ex vivo, but also had lower concentrations of monocyte chemoattractant protein-1 (MCP-1) and eotaxin. Preincubation of the cells with alpha-GOS failed to affect the migration to peritoneal lavage fluid. We propose that dietary alpha-GOS reduces cell infiltration in allergic peritonitis by reducing antigen-induced elicitation of MCP-1 and eotaxin in mice.

생쥐의 Sarcoma 180에 대한 금목이(Tremella aurantialba) 자실체 추출 조다당류의 항암 및 면역조절 효과

금목이의 자실체로부터 중성염용액, 열수 및 메탄올을 이용하여 조다당류를 추출하였다. 세포독성 실험결과, 각각의 조다당류는 <TEX>$2000\;{\mu}g/ml$</TEX>의 농도에서 마우스 대식세포 RAW 309 Cr.1와 육종암세포 Sarcoma 180에 대하여 세포독성은 나타나지 않았다. Sarcoma 180가 접종된 ICR mice에 각각의 조다당류를 주사하여 평균 수명 연장 효과를 조사한 결과 실험군은 수명이 대조군에 비해 각각 <TEX>$11.1{\sim}66.7%$</TEX> 연장되었다. 메탄올으로 추출한 조다당류를 <TEX>$200\;{\mu}g/ml$</TEX> 투여한 생쥐의 B 임파구 alkaline phosphatase의 활성은 대조군에 비해 약 1.16배 증가하는 것으로 나타났다. 열수 추출 조다당류를 투여한 생쥐의 복강의 총 세포 수는 대조군에 비하여 최고 1.42배 증가하였으며, 혈액 중 백혈구의 수도 대조군에 비하여 약 2.87배 증가하였다. 또한 면역과 관련된 장기인 간 비장 및 흉선의 무게는 대조군에 비하여 소폭 증가하였다. Tremella aurantialba, one of edible and medicinal mushrooms belonging to Tremellaceae of Basidiomycota, has been known to have outstanding curing effects on coughing, tracheitis and hypertension of humans and antitumor activity on the sarcoma 180 and Erhrlich carcinoma of mice. Neutral saline soluble (0.9% NaCl), hot water soluble and methanol soluble substances (hereinafter referred to Fr. NaCl, Fr. HW and Fr. MeOH, respectively) were extracted from fruiting body of the mushroom. In vitro cytotoxicity tests showed that all crude polysaccharides extracted from the fruiting body were not cytotoxic against cancer cell lines such as RAW 309 CR.1 and Sarcoma 180 at the concentration of <TEX>$2000\;{\mu}g/ml$</TEX>. Intraperitoneal injection with crude polysaccharides exhibited life prolongation effect of <TEX>$11.1{\sim}66.7%$</TEX> in mice inoculated with Sarcoma 180. Fr. MeOH improved the immunopotentiation activity of B lymphocyte by increasing the alkaline phosphatase activity by 1.16 folds at the concentration of <TEX>$200\;{\mu}g/ml$</TEX>. In case of Fr. HW, the numbers of peritoneal exudate cells and circulating leukocytes were increased by 1.42 and 2.87 folds, respectively.

장미무당버섯(Russula rosacea)의 자실체에서 추출한 조다당류의 생쥐 Sarcoma 180에 대한 면역증강 및 항암 효과

Russula rosacea, one of edible and medicinal mushroom belonging Agaricales of Basidiomycota, has been known to have good inhibitory effects on sarcoma 180 and Ehrlich carcinoma of mice. Neutral saline soluble (0.9% NaCl), hot water soluble and methanol soluble substances (hereinafter referred to Fr. NaCl, Fr. HW and Fr. MeOH, respectively) were extracted from fruiting body of the mushroom. In vitro cytotoxicity tests, crude polysaccharides were not cytotoxic against cancer cell lines such as Sarcoma 180, HepG2, HT-29 and NIH3T3 at the concentration of . Intraperitoneal injection with crude polysaccharides exhibited life prolongation effect of in mice previously inoculated with Sarcoma 180. Fr. HW improved the immuno-potentiation activity of B lymphocyte by increasing the alkaline phosphatase activity by 6.8 fold compared with control at the concentration of . In case of Fr. NaCl, the numbers of peritoneal exudate cells and circulating leukocytes were increased by 6 and 2.6 folds at the concentration of 50 mg/kg, respectively. Therefore, the antitumor effect against mice Sarcoma 180 by Russula rosacea could be due to immunomodulating activity.

대향고(Lentinus giganteus)의 자실체에서 추출한 조다당류가 생쥐의 Sarcoma 180에 미치는 항암 및 면역 증강효과

본 연구는 대향고의 자실체로부터 중성염용액, 열수 및 메탄올을 이용하여 조다당류를 추출하여 이를 ICR mice에 주사하여 항암 및 면역증강 효과를 조사하였다. Sarcoma 180, HepG2, RAW 309 Cr.1 등의 세포주에 대한 독성 실험결과, 각각의 세포주는 <TEX>$10{\sim}2000\;{\mu}g/ml$</TEX> 조다당류 농도에서 세포독성을 나타내지 않았으나, NIH3T3 세포의 경우에는 열수와 중성염용액에서 추출한 조다당류 <TEX>$10{\sim}2000\;{\mu}g/ml$</TEX>의 농도에서 약간의 독성을 나타내었고, 메탄올에서 추출한 조다당류 <TEX>$2000\;{\mu}g/ml$</TEX>의 농도에서는 독성을 나타내지 않았다. 각각의 조다당류가 투여된 실험군은 대조군에 비해 수명이 각각 <TEX>$14.3{\sim}67.5%$</TEX> 연장되었다. 중성염용액으로 추출한 조다당류는 B 임파구의 alkaline phosphatase 활성을 <TEX>$50{\sim}200\;{\mu}g/ml$</TEX>의 농도에서 대조군에 비해 약 <TEX>$1.53{\sim}1.68$</TEX>배 내외의 증가율을 나타냈다. 중성염추출 조다당류를 50 mg/kg body weight의 농도로 투여한 생쥐의 총 복강 세포 수는 대조군에 비하여 최고 7.7배 증가하였으며, 메탄올 추출물을 50 mg/kg body weight의 농도로 투여하였을 때 혈액 중 백혈구의 수도 대조군에 비하여 약 1.6배 증가하였다. 또한 면역에 관련된 장기인간, 비장 및 흉선의 체중이 대조군에 비하여 소폭으로 증가된 것을 확인하였다. 따라서 대향고 자실체에서 추출한 조다당류의 Sarcoma 180에 대한 항암작용은 이들 조다당류의 면역 증강효과에 의한 것으로 사료된다. Lentinus giganteus, one of edible and medicinal mushroom belongs to Pleurotaceae of Agaricales, has been known to contain some inhibitive substances on Sarcoma 180 and curative effect on high blood pressure. Neutral saline soluble (0.9% NaCl), hot water soluble and methanol soluble substances (hereinafter referred to Fr. NaCl, Fr. HW and Fr. MeOH, respectively) were extracted from fruiting body of the mushroom. In vitro cytotoxicity tests, crude polysaccharides were not cytotoxic against cancer cell lines such as Sarcoma 180 and HepG2 at the concentration of <TEX>$10{\sim}2,000\;{\mu}g/ml$</TEX>, but crude polysaccharides from Fr. NaCl was toxic to NIH3T3 at the concentration of <TEX>$10{\sim}2,000\;{\mu}g/ml$</TEX>. Intraperitoneal injection with crude polysaccharides showed life prolongation effect of <TEX>$14.3{\sim}67.5%$</TEX> in mice previously inoculated with Sarcoma 180, respectively. Fr. NaCl exhibited the immuno-potentiating activity of B lymphocyte by increasing the alkaline phosphatase activity by <TEX>$1.53{\sim}1.68$</TEX> folds compared with control at the concentration of <TEX>$50{\sim}200\;{\mu}g/ml$</TEX>, respectively. The numbers of peritoneal exudate cells and circulating leukocytes were increased by 7.7 and 1.6 folds by injecting Fr. NaCl and Fr. MeOH into the mice at the concentration of 50 mg/ml body weight, respectively.

민긴뿌리버섯(Oudemansiella radicata)의 자실체로부터 추출한 조다당류의 항암 및 면역 활성 효과에 관한 연구

민긴뿌리버섯은 송이버섯과에 속하는 버섯으로 예로부터 식용은 물론 항암, 고혈압 및 진균감염증의 치료에 널리 이용해온 식의약용 버섯이다. 민긴뿌리버섯의 자실체로부터 중성염용액, 열수 및 메탄올을 이용하여 조다당류를 추출하여 Sarcoma 180에 접종된 ICR mice에 주사하여 수명연장 및 항암효과를 조사하였다. 세포독성 실험결과, 각각의 세포는 <TEX>$10{\sim}1000\;{\mu}g/ml$</TEX> 추출물 농도에서 70% 내외의 생존율을 보여 세포독성을 나타내지 않았다. 각각의 조다당류가 투여된 실험군이 대조군에 비해 평균수명이 각각 <TEX>$42.9{\sim}66.7%$</TEX> 연장되었다. 중성염용액 추출물은 B 임파구의 alkaline phosphatase 활성을 대조군과 LPS군에 비해 약 <TEX>$1.4{\sim}3$</TEX>배 내외의 증가율을 보였다. 총 복강 세포수도 대조군에 비하여 최고 3.5배 정도 증가하였으며, 혈액 중 백혈구의 수도 대조군에 비하여 약 2.5배 증가하였다. 그리고 면역에 관련된 장기인 간, 비장 및 흉선의 체중이 대조군에 비하여 증가된 것을 확인하였다. Oudemansiella radicata, edible and medicinal mushroom belonging to Agaricales of Basidiomycota, has been known to exhibit outstanding curative effects on the fungal infection and hypertension caused by high blood pressure. Neutral saline soluble (0.9% NaCl), hot water soluble and methanol soluble substances (hereinafter referred to Fr, NaCl, Fr. HW and Fr. MeOH, respectively) were extracted from fruiting body of the mushroom. In vitro cytotoxicity test showed that Fr. NaCl was not cytotoxic against NIH3T3 and Sarcoma 180 at the concentration of <TEX>$10{\sim}1,000\;{\mu}g/ml$</TEX>. Intraperitoneal injection with Fr. NaCl exhibited antitumor activity with life prolongation effect of <TEX>$42.9{\sim}66.7%$</TEX> in mice inoculated with Sarcoma 180. Fr. NaCl improved the immunopotentiating activity of B lymphocyte by increasing the alkaline phosphatase activity by <TEX>$1.4{\sim}3$</TEX> folds compared with controlled and LPS groups, respectively. Intraperitoneal injection with Fr. MeOH increased the numbers of peritoneal exudate cells and circulating leukocytes by 3.5 and 2.5 folds, respectively. Therefore, the antitumor effect exhibited on mouse Sarcoma 180 cells was likely due to immune-modulating activity of crude polysaccharides extracted from fruiting body of O. radicata.

뽕나무버섯부치(Armillaris tabescens)의 자실체에서 추출한 조다당류의 생쥐 Sarcoma 180에 대한 항암 및 면역증강 효과

Armillaria tabescens, one of edible and medicinal mushrooms belonging to Agaricales of Basidiomycota, has been known to have outstanding curative effects on chronic hepatitis and cholecystitis and inhibitory effects on the sarcoma 180 and Erhrlich carcinoma of mice. Neutral saline soluble (0.9% NaCl), hot water soluble and methanol soluble substances (hereinafter referred to Fr, NaCl, Fr. HW and Fr, MeOH, respectively) were extracted from fruiting body of the mushroom. In vitro cytotoxicity tests showed that crude polysaccharides were not cytotoxic against cancer cell lines such as NIH3T3 and Sarcoma 180 at the concentration of . Intraperitoneal injection with crude polysaccharides exhibited life prolongation effect of in mice inoculated with Sarcoma 180, respectively. Fr. NaCl improved the immunopotentiation activity of B lymphocyte by increasing the alkaline phosphatase activity by folds, respectively. In case of Fr, NaCl, the numbers of peritoneal exudate cells and circulating leukocytes were increased by 9 and 1.9 folds, respectively.

뽕나무버섯(Armillaria mellea)의 자실체에서 추출한 조다당류가 생쥐의 Sarcoma 180에 미치는 억제효과

뽕나무버섯은 담자균문 송이과에 속하며 예로부터 항암 효과를 나타내는 식용버섯으로 알려져 있다. 뽕나무버섯 자실체로부터 중성염용액, 열수 및 메탄올 추출물을 분리하여 생쥐에 대한 항암 및 면역증강효과를 조사하였다. Sarcoma 180 복수암 세포를 주사하고 뽕나무버섯 조다당류를 투여한 실험군의 생쥐가 조다당류를 투여하지 않은 대조군의 생쥐에 비해 생명연장효과가 67.5%로 매우 높게 나타났다. 세포독성 실험결과, NIH3T3와 Sarcoma 180 세포주는 중성염용액, 열수 및 메탄올 추출물 <TEX>$1000{\mu}g/ml$</TEX>의 농도에서 배양하였을때 70% 내외의 생존률을 보였으며, 각 농도 구간에서도 세포독성을 나타내지 않았다. 중성염용액 추출물을 투여한 생쥐의 B 임파구 alkaline phosphatase 활성은 대조군에 비하여 약 3.0배 내외의 증가율을 보였으며, 총 복강 세포수도 대조군에 비해 최고 3.5배 가량 증가하였으며, 혈액 중 백혈구의 수도 대조군에 비하여 약 2.5배 증가하였다. 그리고 면역에 관련된 장기인 간, 비장 및 흉선의 체중도 대조군에 비하여 증가한 것을 확인하였다. 따라서 뽕나무버섯의 조다당류 추출물을 투여한 생쥐는 Sarcoma 180에 대한 항암 효과는 면역 활성의 증가에 의한 것으로 사료된다. Armillaria mellea, one of edible and medicinal mushroom belonging to Tricholomataceae of Basid-iomycota, has been known to have outstanding inhibitive effects on the sarcoma 180 and Erhrlich carcinoma of mice. Neutral salt soluble (0.9% NaCl), hot water soluble and methanol soluble substances (hereinafter referred to Fr. NaCl, Fr, HW and Fr. MeOH, respectively) were extracted from the mushroom. In vitro cytotoxicity tests, crude polysaccharide were not cytotoxic against cancer cell lines such as NIH3T3 and Sarcoma 180 at the concentration of <TEX>$1000{\mu}g/ml$</TEX>. Intraperitoneal injection with crude polysaccharides exhibited life prolongation effect of <TEX>$60{\sim}67.5%$</TEX> in mice inoculated with Sarcoma 180, respectively. Fr, NaCl improved the immunopotentiation activity of B lymphocyte by increasing the alkaline phosphatase activity by <TEX>$1.8{\sim}3.0$</TEX> folds, respectively. In case of Fr. NaCl, the numbers of peritoneal exudate cells and circulating leukocytes were increased by 10 and 2 folds, respectively.

Immunostimulant activity of an enzymatic protein hydrolysate from green microalga Chlorella vulgaris on undernourished mice

This study examined the effects of oral administration of an enzymatic protein hydrolysate from green microalga Chlorella vulgaris (Cv-PH) on the recovery of both innate and specific immune responses of undernourished Balb/c mice after a 3-day fasting period. Cv-PH was prepared by hydrolysis of ethanol-extracted cell biomass with pancreatin (20 AU/g) at pH 7.5 and 45 °C for 4 h. The treatment with Cv-PH (500 mg/kg) for 8 days provided benefits in terms of haemopoiesis, as judged by the recovery of bone marrow cellularity and the leukocyte counts in peripheral blood, particularly the lymphocyte pool, which increased up to 128% compared to control animals. Starved mice treated with Cv-PH showed a higher number of peritoneal exudate cells and the macrophage activation was demonstrated by the enhancement in glucose consumption and acid phosphatase activities relative to non-supplemented mice. The increased carbon clearance in peripheral blood suggested the stimulation of mononuclear phagocytic system. Cv-PH also stimulated both humoral and cell mediated immune functions positively, such as T-dependent antibody response and the reconstitution of delayed-type hypersensitivity response (DTH). These findings indicate that Chlorella protein hydrolysate can be used for developing physiologically functional foods with immunopotentiating activity.

Antiapoptotic and immunomodulatory effects of chlorophyllin

Chlorophyllin (CHL) was earlier shown to reduce the level of intracellular ROS and apoptosis induced by ionizing radiation and 2,2′-azobis(2-propionimidinedihydrochloride) (AAPH). In the present studies, the effect of CHL on radiation-induced immunosuppression and modulation of immune responses in mice was examined. Chlorophyllin inhibited the in vitro lymphocyte proliferation induced by concanavalin A (Con A) in a dose dependent manner at doses ≥50 μM. At lower doses (10 μM) CHL significantly inhibited activation induced cell death (AICD) in Con A stimulated spleen cells. Spleen cells obtained from CHL treated mice showed an inhibition of response to Con A depending on dose of CHL and the time after its administration. Spleen cells obtained from CHL treated mice (24 h) showed lower inhibition of response to Con A following in vitro (5 Gy) as well as whole body irradiation (2 Gy). The expression of antiapoptotic genes bcl-2 and bcl-x L was up-regulated in these cells. Chlorophyllin treatment of mice led to splenomegaly and increase in the number of peritoneal exudate cells (PEC). The numbers of T cells, B cells and macrophages in the spleen were also increased. Increased phagocytic activity was seen in PEC obtained from CHL treated mice. Most importantly, CHL administration to mice immunized with sheep red blood cells (SRBC) augmented both humoral and cell-mediated immune responses.

Recovery of age‐dependent immunological deterioration in old mice by thyroxine treatment

On the basis that multiple interactions exist between thyroid hormones and immune system, and ageing is accompanied by changes in thyroid hormone secretion, it seems possible that thyroid hormones may be involved in the age-related immune dysfunction. The present study was conducted to evaluate in vivo and in vitro effects of thyroxine (T(4)) treatment on both cell-mediated and humoral immune responses of aged mice. In a trial to improve age-associated immune dysfunction, T(4) (0.2, 1.0 and 5.0 microg) was subcutaneously supplemented to BALB/c mice (over 18 months old) for 30 consecutive days. The present results showed that exogenous treatment of aged mice with T(4) was associated with a marked increase in serum T(4) level, and the total number of peripheral blood leukocytes as well as the total cellularity of thymus, spleen, peripheral lymph nodes (PLNs), mesenteric lymph nodes (MLNs) and bone marrow (BM). T(4) treatment also caused a significant increase in the total and differential numbers of peritoneal exudate cells (PECs), while it caused a slight increase in macrophages' phagocytic activity of PEC. Moreover, T(4) treatment elicited a statistically significant increase in both plaque-forming cell and rosette-forming cell responses. In vitro results showed that the addition of T(4) at concentrations of 0.001, 0.005 and 0.025 microg/well substantially potentiated the ability of splenocytes from aged mice to proliferate in the presence of concanavalin-A mitogen. Histological examination of thymuses from T(4)-treated aged mice revealed that the cortex was preferentially enlarged and repopulated with immature thymocytes. The present study postulates that thyroid hormones may be involved in the observed decrease in the immune responsiveness during ageing, and that T(4) treatment to aged mice is able to restore the age-related decline of the immune efficiency.

Cytocidal Effect ofStreptococcus pyogeneson Mouse Neutrophils In Vivo and the Critical Role of Streptolysin S

We analyzed the in vivo dynamics of peritoneal exudate cells (PECs) in mice injected with group A streptococcus (GAS). A live low-virulence strain, as well as heat-killed low- and high-virulence strains, significantly increased the number of PECs (primarily neutrophils), whereas a live high-virulence strain did not. When coinjected with thioglycollate, the live high-virulence strain, as well as most other GAS strains, suppressed the ability of thioglycollate to induce neutrophil exudation. This suppression was due to a cytocidal effect of GAS on exuded neutrophils rather than an inhibition of neutrophil migration. In addition, GAS enhanced the apoptosis of neutrophils. These cytocidal effects were significantly reduced by the deletion of functional streptolysin S from GAS. Our findings suggest that, in addition to the production of antiphagocytic factors and survival inside phagocytes, GAS uses a more aggressive method--the elimination of neutrophils--to evade the host's innate immune system.