Number Of Spleen Cells Research Articles

SB431542, An Inhibitor of TGF-β1 Activin Receptor-Like Kinases, Improves the Natural History of Myelofibrosis In Gata1low Mice

AbstractAbstract 462The marrow microenvironment in primary myelofibrosis and mouse models of the disease is characterized by increased levels of cytokines which regulate hematopoiesis including CXCL12, BMP4, VEGF and TGF-β. The observation that TGF-βnull myelofibrotic stem cells fail to transmit the disease by transplantation (Chagraoui et al, Blood 100:3495, 2002) has established an important role for TGF-β in disease development. Mice carrying the hypomorphic Gata1low mutation in which the enhancer that drives gene expression in megakaryocytes (MK) is deleted also develop myelofibrosis with age (Vannucchi et al, Blood 2002;100:1123). To clarify the role of TGF-β in development of myelofibrosis in the Gata1low mouse model, the levels of this factor in plasma and marrow of the mutant mice were measured. Gata1low mice express normal levels of TGF-β in plasma (1.8±0.7 vs 2.1±0.4 ng/mL) and levels of TGF-β mRNA (2.9±0.5 vs 1.5±0.3 arbitrary units, p<0.01) and protein (1.9±0.3 vs 0.86±0.2 ng/mL, p<0.01) only 2-times greater than normal in marrow. However, by immunoelectron microscopy, patchy TGF-β deposits associated with collagen fibers were observed in the marrow microenvironment of mutant mice (<3 vs >700 particles/field in wild-type and Gata1low marrow, respectively) indicating that fibrosis, by concentrating TGF-β locally, may contribute to disease progression also in Gata1low mice. To evaluate whether inhibition of TGF-β signaling would ameliorate myelofibrosis in this animal model, Gata1low mice were treated with SB431542 (C22H16N4O3, MW= 384.4), an inhibitor of TGF- β1/activin receptor-like kinases recently demonstrated to prevent renal fibrosis in mice (Petersen et al, Kidney Int 73:705, 2008.). Six males (7-9 months) and 6 females (12 months) were treated with SB431542 as described for renal fibrosis (see Figure). Equivalent numbers of mice treated with vehicle were used as control. Treatment was well tolerated (no deaths) and the SB43542-treated mice were easily recognized by being more active and with shinier coats. At the end of the 4th cycle, mice were sacrificed and analyzed for disease progression. The results were as follows: Blood:SB43542-treatment did not affect hematocrit levels (43.2±1.2 vs 41.3±0.9 in SB43542- and vehicle-treated mice, respectively), increased platelets numbers [0.34(±0.03)×106/μL vs 0.2(±0.009)×106/μL, p<0.01] but platelets remained larger than normal, reduced white blood cell counts [5.2(±0.19)×103/μL vs 6.5(±0.3)×103/μL, p<0.01] and frequency of poikilocytes (1 every 4–5 fields vs >4/field). Also, progenitor cell trafficking was not reduced (CD34posCD117pos cells: 1.45 vs 1.2% colony forming cells: 10.8±2.4 vs 8.0±0.1 CFC/μL). Marrow:Treatment increased total cell number [18.0(±0.7) ×106 vs 8.2(±0.4) ×106/femur, p<0.01] and frequency of erythroid cells (20.5±2.5 vs 13.2±0.7%, p<0.01) but not of MK (40.2±5.7 vs 38.7±0.9%) in the femur. However, fibrosis and microvessel density were reduced (Gomori-Silver and CD34 staining). Increased Mallory staining of bones was observed but the femur became resistant to fracture, suggesting that overall bone structure improved. Spleen:SB43542-treatment reduced spleen weight (0.2±0.6 vs 0.45±0.05 gr, p<0.01) and cell numbers [265(±30)×106 vs 385(±5)×106 cells, p<0.01]. Therefore, although the frequency of erythroid cells and MK in the organ remained high, overall hematopoiesis in spleen was reduced. Liver:SB43542-treatment restored the morphological appearance of the liver and reduced the frequency of MK (5.9±0.7 vs 20.4±4.7, p<0.01). These improvements were likely not due to an anti-inflammatory effect of the drug because parallel treatments with dexamethasone did not modify disease progression in Gata1low mice. Conclusion:SB43542-treatment reduced the myelofibrotic traits expressed by Gata1low mice, confirming that increased TGF-β1 levels play an important role in disease manifestations in this animal model. We have previously published that Aplidin treatment restores the hematopoietic stem cell properties of Gata1low mice (Verrucci et al, J Cell Physiol,2010, May20, Epub ahead of print). The observation that SB43542-treatment primarily reduced microenvironmental abnormalities suggests that the two drugs may have synergistic effects in the treatment of myelofibrosis. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Non-Hematopoietic Stromal Cells Sense Toll-Like Receptor 4 Agonists and Consequently Enhance Myelopoiesis.

AbstractAbstract 2583Hematopoiesis is tightly regulated by growth factors acting on stem and progenitor cells (HSPCs) in the bone marrow. During systemic infections cytokines are elevated in serum, myelopoiesis is enhanced, and myeloid CFUs and granulocytes increase in circulation. However, the underlying mechanisms of this “emergency” myelopoietic response have not been defined. Sensing of conserved pathogen-associated products by specialized pattern-recognition receptors such as Toll-like receptors (TLRs) is crucial for rapid responses to infection. Based on the well-known regulatory function of the BM microenvironment, we hypothesized that bone marrow stromal cells (BMSCs) express TLRs and possess all functional properties required to sense microbes and drive emergency myelopoiesis. Human BMSCs expressed Tlr1, Tlr5, and Tlr6 at similar levels and Tlr3 and Tlr4 mRNA at about 2-log higher compared to dendrititc cells (DCs). Stimulation of BMSCs with the TLR4 agonist LPS led to de novo expression of G-csf and Gm-csf, and increased M-csf, Il-6, and Il-11 expression. In line with this, LPS induced production of G-CSF and GM-CSF protein and significantly enhanced the secretion of M-CSF, IL-6, and IL-11. Using LPS-stimulated BMSC culture supernatant in myeloid CFU assays led to a 2.5-fold higher myeloid CFU activity compared to un-stimulated BMSC supernatant. This effect was partly mimicked by adding G-, M-, and GM-CSF to the methylcellulose cultures. Importantly, direct LPS stimulation of CB CD34+ cells had no effect. Furthermore, co-culture of BMSCs and CB CD34+ cells together with LPS for 12 days led to approximate 2-fold higher recovery of immuno-phenotypically primitive CD34+ cells, and retained up to 8-fold more CD34+ cells in divisions 0–3 as compared to LPS-free co-cultures as measured by CFSE dilution. When subjected to cytokine-supplemented myeloid CFU assays or transplanted into newborn RAG2-/- γc-/- mice to evaluate lymphoid differentiation, recovered CD34+ cells from LPS-stimulated BMSC cultures gave rise to the full spectrum of myeloid colonies and T and B cells, respectively, thus proving maintenance of primitive hematopoietic progenitors. To elucidate the in vivo relevance of the findings and to clarify the contribution of stromal vs. hematopoietic cell expressed TLR4, we generated chimeras with TLR4-/- hematopoiesis in a wild-type (WT) background (hematopoietic-TLR4-/-) and WT hematopoiesis in a TLR4-/- background (non-hematopoietic-TLR4-/-). Chimeric, WT, and TLR4-/- mice were injected with LPS and hallmarks of myelopoietic responses such as G-CSF expression, myeloid cell mobilization from the BM, and increased myeloid cell production in the BM was evaluated. Significant G-csf mRNA induction could be observed in the BM of WT and hematopoietic-TLR4-/- mice. To a much lesser, non-significant extent, this effect could be observed also in non-hematopoietic-TLR4-/- mice, while no transcripts were detectable in TLR4-/- mice. Accordingly, serum G-CSF levels significantly increased 10-fold in WT and hematopoietic-TLR4-/- mice after LPS injection, but no increase was detectable in non-hematopoietic-TLR4-/- and TLR4-/- mice. LPS injection also resulted in a significant decrease in BM cellularity accompanied by an increase of spleen cell numbers only in WT and hematopoietic-TLR4-/- mice. Furthermore, Gr-1highCD11blow/+ mature myeloid cells were significantly reduced whereas Gr-1lowCD11blow/+ immature promyelocytes and myelocytes significantly increased (2.5-fold) in the BM of WT and hematopoietic-TLR4-/- mice. In contrast, similar changes in cellular composition could not be observed in TLR4-/- and non-hematopoietic-TLR4-/- mice, while a small, but still significant 1.25-fold increase in immature Gr-1lowCD11blow/+ cells was detectable in non-hematopoietic-TLR4-/- mice. Finally, inflammation-induced Sca-1 upregulation on HSPCs and increasing frequencies of GMPs were only observed in WT and hematopoietic-TLR4-/- mice. Collectively, our in vitro data demonstrate that human BMSCs are able to sense pathogens and stimulate emergency myelopoiesis but also prevent loss of HSPCs by enhancing their maintenance. Importantly, in vivo signaling via non-hematopoietic cell-expressed TLR4 is sufficient and is the main mechanism regulating both the release of mature myeloid cells from and the enhanced myeloid cell production in the bone marrow during systemic challenges. Disclosures:No relevant conflicts of interest to declare.

The eIF2 Kinase GCN2 Is Essential for the Murine Immune System to Adapt to Amino Acid Deprivation by Asparaginase1–3

Amino acid starvation by asparaginase (ASNase) enhances phosphorylation of eukaryotic initiation factor 2 (eIF2) by general control nonderepressible 2 (GCN2) kinase, leading to reduced global mRNA translation rates. This conserves energy and allows cells time to reprogram stress-related gene expression to alleviate cell injury. This study addressed the importance of GCN2 for the immune system to adapt to amino acid starvation by ASNase. GCN2+/+ and GCN2−/− mice were injected once daily with ASNase or saline for up to 7 d. In both thymus and spleen, activation of amino acid stress response genes to ASNase, such as asparagine synthetase and CAAT enhancer binding protein homologous protein, required GCN2. ASNase reduced food intake and body weight in both genotypes, but spleen and thymus wet weights and total cell numbers in thymus, spleen, bone marrow, and mesenteric lymph nodes were less in GCN2−/− mice treated with ASNase (genotype x ASNase, P < 0.05). In the thymus, GCN2−/− mice treated with ASNase demonstrated enhanced apoptosis and fewer cells in all subpopulations examined (CD3+, CD4–8-, CD4+8+, CD4+8-, CD4–8+) compared with GCN2+/+ mice treated with ASNase (genotype x ASNase, P < 0.05). In the spleen, GCN2 deletion magnified ASNase-induced reductions in CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD11b+ leukocytes (genotype x ASNase, P < 0.05). These results indicate that loss of GCN2 enhances immunosuppression by ASNase and that this eIF2 kinase is broadly required for amino acid stress management in the immune system.

Prenatal Exposure to Non-ionizing Radiation: Effects of WiFi Signals on Pregnancy Outcome, Peripheral B-Cell Compartment and Antibody Production

During embryogenesis, the development of tissues, organs and systems, including the immune system, is particularly susceptible to the effects of noxious agents. We examined the effects of prenatal (in utero) exposure to WiFi signals on pregnancy outcome and the immune B-cell compartment, including antibody production. Sixteen mated (plug-positive) female mice were assigned to each of the following groups: cage control, sham-exposed and microwave-exposed (WiFi signals at 2.45 GHz, whole body, SAR 4 W/kg, 2 h/day, 14 consecutive days starting 5 days after mating). No effects due to exposure to WiFi signals during pregnancy on mating success, number of newborns/mother and body weight at birth were found. Newborn mice were left to grow until 5 or 26 weeks of age, when immunological analyses were performed. No differences due to exposure were found in spleen cell number, B-cell frequency or antibody serum levels. When challenged in vitro with LPS, B cells from all groups produced comparable amounts of IgM and IgG, and proliferated at a similar level. All these findings were consistently observed in the female and male offspring at both juvenile (5 weeks) and adult (26 weeks) ages. Stress-associated effects as well as age- and/or sex-related differences were observed for several parameters. In conclusion, our results do not show any effect on pregnancy outcome or any early or late effects on B-cell differentiation and function due to prenatal exposure to WiFi signals.

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.

Absence of mitochondrial uncoupling protein 1 affects apoptosis in thymocytes, thymocyte/T-cell profile and peripheral T-cell number

Our laboratory has previously demonstrated the presence of constitutively expressed mitochondrial uncoupling protein 1 in mouse thymocytes. In our endeavours to understand the role of mitochondrial uncoupling protein 1 in thymocyte function, we compared cell profiles in thymus and spleen of wild-type with those of UCP 1 knock-out mice, which in turn led to comparative investigations of apoptotic potential in thymocytes from these mice. We demonstrate that spleen cell numbers were reduced ∼3-fold in UCP 1 knock-out mice compared to wild-type mice. We record a halving of CD8 single positive cell numbers in thymus with a significant incremental increase in CD4/CD8 double positives cell numbers in the thymus of UCP 1 knock-out mice compared to wild-type mice. These data are mirrored by an approximate halving of CD8 single positive cell numbers and a doubling of CD4/CD8 double positive cell numbers in the spleen of UCP 1 knock-out mice compared to wild-type mice. These differences are most probably explained by our observations of decreased apoptotic potential and higher ATP levels in thymocytes of UCP 1 knock-out mice when compared to wild-type controls. We conclude that constitutively expressed UCP 1 is a factor in determining T-cell population selection in mice.

Inhibition of In Vivo HIV Infection in Humanized Mice by Gene Therapy of Human Hematopoietic Stem Cells with a Lentiviral Vector Encoding a Broadly Neutralizing Anti-HIV Antibody

Due to the inherent immune evasion properties of the HIV envelope, broadly neutralizing HIV-specific antibodies capable of suppressing HIV infection are rarely produced by infected individuals. We examined the feasibility of utilizing genetic engineering to circumvent the restricted capacity of individuals to endogenously produce broadly neutralizing HIV-specific antibodies. We constructed a single lentiviral vector that encoded the heavy and light chains of 2G12, a broadly neutralizing anti-HIV human antibody, and that efficiently transduced and directed primary human B cells to secrete 2G12. To evaluate the capacity of this approach to provide protection from in vivo HIV infection, we used the humanized NOD/SCID/gamma(c)(null) mouse model, which becomes populated with human B cells, T cells, and macrophages after transplantation with human hematopoietic stem cells (hu-HSC) and develops in vivo infection after inoculation with HIV. The plasma of the irradiated NOD/SCID/gamma(c)(null) mice transplanted with hu-HSC transduced with the 2G12-encoding lentivirus contained 2G12 antibody, likely secreted by progeny human lymphoid and/or myeloid cells. After intraperitoneal inoculation with high-titer HIV-1(JR-CSF), mice engrafted with 2G12-transduced hu-HSC displayed marked inhibition of in vivo HIV infection as manifested by a profound 70-fold reduction in plasma HIV RNA levels and an almost 200-fold reduction in HIV-infected human cell numbers in mouse spleens, compared to control hu-HSC-transplanted NOD/SCID/gamma(c)(null) mice inoculated with equivalent high-titer HIV-1(JR-CSF). These results support the potential efficacy of this new gene therapy approach of using lentiviral vectors encoding a mixture of broadly neutralizing HIV antibodies for the treatment of HIV infection, particularly infection with multiple-drug-resistant isolates.

Estradiol and G1 Reduce Infarct Size and Improve Immunosuppression after Experimental Stroke

Reduced risk and severity of stroke in adult females is thought to depend on normal endogenous levels of estrogen, a well-known neuroprotectant and immunomodulator. In male mice, experimental stroke induces immunosuppression of the peripheral immune system, characterized by a reduction in spleen size and cell numbers and decreased cytokine and chemokine expression. However, stroke-induced immunosuppression has not been evaluated in female mice. To test the hypothesis that estradiol (E2) deficiency exacerbates immunosuppression after focal stroke in females, we evaluated the effect of middle cerebral artery occlusion on infarct size and peripheral and CNS immune responses in ovariectomized mice with or without sustained, controlled levels of 17-beta-E2 administered by s.c. implant or the putative membrane estrogen receptor agonist, G1. Both E2- and G1-replacement decreased infarct volume and partially restored splenocyte numbers. Moreover, E2-replacement increased splenocyte proliferation in response to stimulation with anti-CD3/CD28 Abs and normalized aberrant mRNA expression for cytokines, chemokines, and chemokine receptors and percentage of CD4(+)CD25(+)FoxP3(+) T regulatory cells observed in E2-deficient animals. These beneficial changes in peripheral immunity after E2 replacement were accompanied by a profound reduction in expression of the chemokine, MIP-2, and a 40-fold increased expression of CCR7 in the lesioned brain hemisphere. These results demonstrate for the first time that E2 replacement in ovariectomized female mice improves stroke-induced peripheral immunosuppression.

Effect of acute whole-body proton irradiation on hematological and intestinal variables in mice

Female C57BL/6 mice were exposed to a single dose of protons or 60Co gamma rays at 20 mGy and 2 Gy, which represent the recommended limit for annual occupational exposure and the daily fractional dose for patients undergoing radiotherapy, respectively. A control group was not exposed to radiation. The number of peripheral blood, bone marrow, and spleen cells was assessed 24 h after radiation to investigate the marrow suppression. The number of lymphocytes and neutrophils in peripheral blood was significantly lower in mice exposed to the higher dose of radiation than in the nonirradiated mice. However, at a low dose of radiation, there were no discernable hematological effects. The frequency of micronuclei in bone marrow cells was increased only after the high dose of gamma radiation. Radiation-induced intestinal apoptosis was measured using terminal deoxynucleotide transferase labeling assay. The magnitude of damage was greater after gamma irradiation than after proton irradiation at the same dose. Among the systemic biological end point to predict whole body radiation effect, the apoptosis of intestinal crypts may be the most sensitive parameter to provide information even at low dose of radiation.

Combined role of extracellular matrix and chemokines on peripheral lymphocyte migration in growth hormone transgenic mice

Previous evidence indicated that growth hormone (GH) modulates cell migration in the thymus, and that extracellular matrix and chemokines are involved. Herein, we studied migration of peripheral lymphocytes derived from spleen and lymph nodes of GH-transgenic (GH-Tg) mice. We initially found that the relative cell numbers (normalized per gram of body weight) in lymph nodes and spleens from GH-Tg were higher at all ages tested (2–3, 7 and 12months), as compared to wild type age-matched controls. Functionally, we found that lymphocyte migration triggered by laminin or fibronectin was enhanced in cells from GH-Tg versus control mice, independent of the organ from which the cells were derived (as ascertained in young adult animals). However, such an enhancement in migration was statistically significant only for CD4+ and CD8+ T cells from mesenteric lymph nodes. Migration of lymphocytes from mesenteric lymph nodes of GH-Tg mice, triggered by the chemokine CXCL12, in conjunction with laminin or fibronectin, was enhanced compared to lymphocytes from control mice. Rather surprisingly, the membrane levels of the corresponding extracellular matrix or chemokine receptors in peripheral lymphoid organs of GH-Tg mice did not necessarily correlate with the changes seen in migratory responses. In conclusion, our data show for the first time that GH alters lymphocyte migration in the periphery of the immune system. Considering that GH is used as an adjuvant therapeutic agent in immunodeficiencies, including AIDS, the concepts defined herein provide relevant background knowledge for future GH-related immune interventions.

Acidic polysaccharide of Panax ginseng as a defense against small intestinal damage by whole-body gamma irradiation of mice

An acidic polysaccharide of Panax ginseng (APG), ginsan, has been reported to protect the hematopoietic system by increasing the number of bone marrow cells and spleen cells. Therefore, we evaluated the ability of APG to protect mice from radiation-induced damage of the small intestine. APG treatment caused the lengthening of villi and a numerical increase of crypt cells in the small intestine at 3.5 days after 7 Gy irradiation compared to irradiated, non-treated controls. In addition, APG significantly inhibited irradiation-induced apoptosis by decreasing the amount of pro-apoptotic p53 and Bax as well as augmenting that of anti-apoptotic Bcl-2 at 24 h after irradiation. These results indicate that APG might be a useful adjunct to therapeutic irradiation as a protective agent for the gastrointestinal tract of cancer patients.

Fusion protein Ag85B-MPT64 190–198-Mtb8.4 has higher immunogenicity than Ag85B with capacity to boost BCG-primed immunity against Mycobacterium tuberculosis in mice

Tuberculosis (TB) remains a major infectious disease worldwide despite chemotherapy and BCG vaccine. The efficacy of the current TB vaccine BCG varies from 0 to 80%. New vaccines that have better protection than BCG or have the capability to boost BCG-primed immunity are urgently needed. We have previously constructed a fusion protein Ag85B-MPT64 190–198-Mtb8.4 (AMM). In this study, we investigated the immunogenicity of the fusion protein AMM in a novel adjuvant of dimethyl-dioctyldecyl ammonium bromide and BCG polysaccharide nucleic acid (DDA–BCG PSN), and its capacity to boost BCG-primed immunity. The anti-Ag85B antibodies IgG1 and IgG2a were determined using ELISA and the number of spleen cells secreting IFN-γ was determined by ELISPOT. In addition, the ability of the subunit vaccine AMM to boost BCG-primed immunity against Mycobacterium tuberculosis was analyzed. The fusion protein AMM induced more effective humoral and cell-mediated immune responses in mice than Ag85B alone. Mice primed with BCG vaccination followed by boosting with AMM produced a stronger immune response and afforded a better protection against M. tuberculosis infection than mice immunized with BCG alone or BCG priming followed by boosting with Ag85B. These findings suggest that AMM is a promising candidate subunit vaccine to enhance the protective efficiency of BCG.

Yersinia enterocolitica Targets Cells of the Innate and Adaptive Immune System by Injection of Yops in a Mouse Infection Model

Yersinia enterocolitica (Ye) evades the immune system of the host by injection of Yersinia outer proteins (Yops) via a type three secretion system into host cells. In this study, a reporter system comprising a YopE-β-lactamase hybrid protein and a fluorescent staining sensitive to β-lactamase cleavage was used to track Yop injection in cell culture and in an experimental Ye mouse infection model. Experiments with GD25, GD25-β1A, and HeLa cells demonstrated that β1-integrins and RhoGTPases play a role for Yop injection. As demonstrated by infection of splenocyte suspensions in vitro, injection of Yops appears to occur randomly into all types of leukocytes. In contrast, upon infection of mice, Yop injection was detected in 13% of F4/80+, 11% of CD11c+, 7% of CD49b+, 5% of Gr1+ cells, 2.3% of CD19+, and 2.6% of CD3+ cells. Taking the different abundance of these cell types in the spleen into account, the highest total number of Yop-injected cells represents B cells, particularly CD19+CD21+CD23+ follicular B cells, followed by neutrophils, dendritic cells, and macrophages, suggesting a distinct cellular tropism of Ye. Yop-injected B cells displayed a significantly increased expression of CD69 compared to non-Yop-injected B cells, indicating activation of these cells by Ye. Infection of IFN-γR (receptor)- and TNFRp55-deficient mice resulted in increased numbers of Yop-injected spleen cells for yet unknown reasons. The YopE-β-lactamase hybrid protein reporter system provides new insights into the modulation of host cell and immune responses by Ye Yops.

Interferon‐α treatment of female (NZW × BXSB)F1 mice mimics some but not all features associated with the Yaa mutation

Male (NZW x BXSB)F(1) mice develop antiphospholipid syndrome (APS) and proliferative glomerulonephritis that is markedly accelerated by the Yaa locus encoding an extra copy of Tlr7. Female (NZW x BXSB)F(1) mice with only 1 active copy of Tlr7 develop late-onset glomerulonephritis but not APS. Because a major function of Toll-like receptor 7 is to induce type I interferons (IFNs), our goal was to determine whether IFNalpha can induce or accelerate the manifestations of systemic lupus erythematosus (SLE) in female (NZW x BXSB)F(1) mice. Eight-week-old female (NZW x BXSB)F(1) mice were injected with a single dose of adenovirus expressing IFNalpha. Mice were monitored for the development of thrombocytopenia and proteinuria. Sera were tested for anticardiolipin and anti-Sm/RNP antibodies. Mice were killed at 17 or 22 weeks of age, and their kidneys and hearts were examined histologically and by immunohistochemistry. Spleen cells were phenotyped, and enzyme-linked immunospot assays for autoantibody-producing B cells were performed. IFNalpha markedly accelerated nephritis and death in female (NZW x BXSB)F(1) mice. A significant increase in spleen cell numbers associated with a striking increase in the number of activated B and T cells was observed. Marginal-zone B cells were retained. IFNalpha-induced increased titers of autoantibodies were observed, but thrombocytopenia was not observed. Cardiac damage was milder than that in male mice. IFNalpha accelerates the development of renal inflammatory disease in female (NZW x BXSB)F(1) mice but induces only mild APS and does not induce thrombocytopenia. The effect of IFNalpha on SLE disease manifestations is strain dependent. These findings are relevant to our understanding of the physiologic significance of the IFN signature.

Immunostimulation and anti-DNA antibody production by backbone modified CpG-DNA

Oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG-DNA) have gained attention as potentially useful therapeutics. However, the phosphorothioate-modified CpG-DNAs (PS-ODN) can induce backbone-related side effects. Here, we compared the immunostimulatory activity of natural phosphodiester CpG-DNA (PO-ODN) from Mycobacterium bovis and PS-ODN in mice. Both PO-ODN and PS-ODN induced production of IL-12. PS-ODN increased spleen weights, spleen cell numbers, and the migration of macrophages into the peritoneal cavity in the mice in a CG sequence-dependent manner. PS-ODN induced anti-PS-ODN antibody production in the mice, and the PS-ODN-specific IgM was cross-reactive with other PS-ODNs in a CG sequence-independent manner. In contrast, PO-ODN did not affect on spleen weights, cell numbers, or IgM production. These results may provide an explanation for the side effects in immunotherapeutic application of PS-ODN. They also suggest that PO-ODN may be more optimal than PS-ODN to enhance innate immune responses without severe side effects.

Lack of Plasma Protein Hemopexin Dampens Mercury-Induced Autoimmune Response in Mice

Several factors affect the autoimmune response, including iron-dependent modulation of T cells. Hemopexin is the plasma protein with the highest binding affinity to heme. It mediates heme-iron recovery in the liver, thus controlling heme-iron availability in peripheral cells. The aim of the present study was to investigate the role of hemopexin in the progress of an autoimmune response. To this end, we chose a mouse model of mercury-induced autoimmunity and evaluated the susceptibility of hemopexin-null mice to mercury treatment compared with wild-type controls. In this study we show that lack of hemopexin dampens mercury-induced autoimmune responses in mice. Hemopexin-null mice produced fewer antinuclear autoantibodies and had reduced deposits of immune complexes in the kidney after mercuric chloride treatment compared with wild-type mice. These features were associated with a reduction in activated T cells and lower absolute B cell number in spleen and impaired IgG1 and IgG2a production. In contrast, in hemopexin-null mice the response to OVA/CFA immunization was maintained. In addition, hemopexin-null mice had reduced transferrin receptor 1 expression in T cells, possibly due to the increase in heme-derived iron. Interestingly, CD4(+)T cells isolated from mercury-treated hemopexin-null mice show reduced IFN-gamma-dependent STAT1 phosphorylation compared with that of wild-type mice. Our data suggest that hemopexin, by controlling heme-iron availability in lymphocytes, modulates responsiveness to IFN-gamma and, hence, autoimmune responses.

Influence of FK506 and cyclosporin A on alloantibody production and lymphocyte activation following blood transfusion

The effect of administration of cyclosporin A (CyA) or the novel macrolide FK506 was investigated in AO rats given DA blood transfusions. CyA (10 mg/kg, orally) or FK506 (1 mg/kg, intramuscularly) administered for 14 days from the time of transfusion effectively inhibited primary anti-MHC class I alloantibody production. This profound inhibitory effect persisted throughout the 2-month investigation period, with little increase in 'secondary' alloantibody production following a challenge injection 28 days after drug withdrawal. Flow cytometric analysis revealed no significant differences in the absolute numbers of W3/25+ (CD4+), OX-8+ (CD8+) or OX-12+ (B lymphocytes), in either the spleen or peripheral blood of transfused compared with normal, untreated animals. However, a small but significant increase in the numbers of splenocytes expressing the activation marker OX-40 (activated CD4+ cells) was observed in transfused animals. Either CyA or FK506 significantly reduced the number of cells expressing OX-39 (interleukin-2 receptors) and OX-40. Treatment of transfused animals with CyA, but not FK506 for 14 days resulted in minor, transient reduction in peripheral blood OX-19+ and W3/25+ cells, while 'sparing' the OX-8+ cells; these changes were not observed in spleens. In contrast, the absolute spleen cell numbers of OX-19+, W3/25+ and OX-8+ cells were significantly reduced in transfused animals given 14 days of FK506 treatment, while the corresponding blood cells were unaffected. Induction of splenic lymphoproliferative responses by the T cell mitogen concanavalin A remained normal in animals receiving transfusion alone or with CyA. In contrast, profound inhibition of mitogenic responses was observed in FK506-treated animals and this inhibitory effect declined gradually following drug withdrawal. No non-specific suppressor cell activity was detected in the spleens of rats given transfusion alone or in CyA or FK506-treated transfused animals.

The effects of CP 17193, an immunosuppressive pyrazaloquinoline, on the development of spontaneous lupus disease in NZBW F1 hybrid mice.

The effects of the immunosuppressive agent CP 17193 on the development of spontaneous lupus disease in female NZBW F1 hybrid mice were investigated. Long term dosing with CP 17193 markedly delayed the onset of mortality but did not extend the long term survival of the mice. CP 17193 significantly inhibited immune complex deposition in the glomeruli of 30- and 35-week-old mice and also reduced the levels of proteinuria in the 35-week-old mice. There was a slight reduction in the levels of circulating antinuclear antibody to ds DNA in CP 17193-treated mice but this was not statistically significant. Studies on immune cell function of 35-week-old mice dosed with CP 17193 showed significant reduction in the total numbers of spontaneous polyclonal antibody producing cells. Analysis of the results revealed these effects to result from a marked reduction in total spleen cell numbers in CP 17193-treated mice. When results were expressed as activity per cell unit the differences between drug-treated and control mice were small. Spleen cells from mice given a shorter dosing schedule of 7 weeks with CP 17193 showed an augmentation of IL-2 production and responsiveness. These results show CP 17193 having interesting selective immunomodulating activity on the immunopathogenesis of spontaneous murine lupus disease. Furthermore, compounds with this profile of activity may have a potential role in the treatment of some autoimmune diseases.

TGF‐β‐induced Foxp3+ regulatory T cells rescue scurfy mice

Scurfy mice have a deletion in the forkhead domain of the forkhead transcription factor p3 (Foxp3), fail to develop thymic-derived, naturally occurring Foxp3+ regulatory T cells (nTreg), and develop a fatal lymphoproliferative syndrome with multi-organ inflammation. Transfer of thymic-derived Foxp3+ nTreg into neonatal Scurfy mice prevents the development of disease. Stimulation of conventional CD4+Foxp3(-) via the TCR in the presence of TGF-beta and IL-2 induces the expression of Foxp3 and an anergic/suppressive phenotype. To determine whether the TGF-beta-induced Treg (iTreg) were capable of suppressing disease in the Scurfy mouse, we reconstituted newborn Scurfy mice with polyclonal iTreg. Scurfy mice treated with iTreg do not show any signs of disease and have drastically reduced cell numbers in peripheral lymph nodes and spleen in comparison to untreated Scurfy controls. The iTreg retained their expression of Foxp3 in vivo for 21 days, migrated into the skin, and prevented the development of inflammation in skin, liver and lung. Thus, TGF-beta-differentiated Foxp3+ Treg appear to possess all of the functional properties of thymic-derived nTreg and represent a potent population for the cellular immunotherapy of autoimmune and inflammatory diseases.

Evaluation of the Immune System in Rats and Mice Administered Linear Ammonium Perfluorooctanoate

Repeated high doses of ammonium perfluorooctanoate (APFO) have been reported to affect immune system function in mice. To examine dose-response characteristics in both rats and mice, male CD rats and CD-1 mice were dosed by oral gavage with 0.3-30 mg/kg/day of linear APFO for 29 days. Anti-sheep red blood cell (SRBC) IgM levels, clinical signs, body weights, selected hematology, and lipid parameters, liver weights, spleen, and thymus weights and cell number, selected histopathology, and serum corticosterone concentrations were evaluated. In rats, linear APFO had no effect on production of anti-SRBC antibodies. Ten and 30 mg/kg/day resulted in systemic toxicity as evidenced by decreases in body weight gain to 74 and 37%, and increases in serum corticosterone levels to 135 and 196% of control, respectively. In mice dosed with 10 and 30 mg/kg/day, marked systemic toxicity and stress were observed, as evidenced by a loss in body weight of 3.8 and 6.6 g, respectively (despite a tripling of liver weight), approximately 230% increase in serum corticosterone, and increases in absolute numbers of peripheral blood neutrophils and monocytes with an accompanying decrease in absolute lymphocyte numbers. Immune-related findings at 10 and 30 mg/kg/day that likely represent secondary responses to the systemic toxicity and stress observed at these doses include: decreased IgM antibody production at 10 (20% suppression) and 30 mg/kg/day (28% suppression); decreased spleen and thymus weights and cell numbers; microscopic depletion/atrophy of lymphoid tissue at 10 (thymus) and 30 mg/kg/day (spleen). In summary, no immune-related changes occurred in rats, even at doses causing systemic toxicity. In mice, immune-related changes occurred only at doses causing significant and profound systemic toxicity and stress.