Influence of Maternal Gestational Treatment with Mycobacterial Antigens on Postnatal Immunity in an Experimental Murine Model

Background

It has been proposed that the immune system could be primed as early as during the fetal life and this might have an impact on postnatal vaccination. Therefore, we addressed in murine models whether gestational treatment with mycobacterial antigens could induce better immune responses in the postnatal life.

Methods/Findings

BALB/c mice were treated subcutaneously (s.c.) at the second week of gestation with antigen (Ag)85A or heparin-binding hemagglutinin (HBHA) in the absence of adjuvant. Following birth, offspring mice were immunized intranasally (i.n.) with the same antigens formulated with the adjuvant cholera toxin (CT) at week 1 and week 4. One week after the last immunization, we assessed antigen-specific recall interferon gamma (IFN-γ) responses by in vitro restimulation of lung-derived lymphocytes. Protection against infection was assessed by challenge with high dose Mycobacterium bovis Bacille Calmette-Guérin (BCG) given i.n. We found that recall IFN-γ responses were higher in the offspring born to the treated mother compared to the untreated-mother. More importantly, we observed that the offspring born to the treated mother controlled infection better than the offspring born to the untreated mother. Since the gestational treatment was done in absence of adjuvant, essentially there was no antibody production observed in the pregnant mice and therefore no influence of maternal antibodies was expected. We hypothesized that the effect of maternal treatment with antigen on the offspring occurred due to antigen transportation through placenta. To trace the antigens, we conjugated fluorescent nanocrystals with Ag85A (Qdot-ITK-Ag85A). After inoculation in the pregnant mice, Qdot-ITK-Ag85A conjugates were detected in the liver, spleen of pregnant females and in all the fetuses and placentas examined.

Conclusion

The fetal immune system could be primed in utero by mycobacterial antigens transported through the placenta.     

Establishment of a New Murine Elastase-Induced Aneurysm Model Combined with Transplantation

Introduction

The aim of our study was to develop a reproducible murine model of elastase-induced aneurysm formation combined with aortic transplantation.

Methods

Adult male mice (n = 6–9 per group) underwent infrarenal, orthotopic transplantation of the aorta treated with elastase or left untreated. Subsequently, both groups of mice were monitored by ultrasound until 7 weeks after grafting.

Results

Mice receiving an elastase-pretreated aorta developed aneurysms and exhibited a significantly increased diastolic vessel diameter compared to control grafted mice at 7 week after surgery (1.11±0.10 mm vs. 0.75±0.03 mm; p≤0,001). Histopathological examination revealed disruption of medial elastin, an increase in collagen content and smooth muscle cells, and neointima formation in aneurysm grafts.

Conclusions

We developed a reproducible murine model of elastase-induced aneurysm combined with aortic transplantation. This model may be suitable to investigate aneurysm-specific inflammatory processes and for use in gene-targeted animals.     

Anti-CD45 Radioimmunotherapy with 90Y but Not 177Lu Is Effective Treatment in a Syngeneic Murine Leukemia Model

Radioimmunotherapy (RIT) for treatment of hematologic malignancies has primarily employed monoclonal antibodies (Ab) labeled with ¹³¹I or ⁹⁰Y which have limitations, and alternative radionuclides are needed to facilitate wider adoption of RIT. We therefore compared the relative therapeutic efficacy and toxicity of anti-CD45 RIT employing ⁹⁰Y and ¹⁷⁷Lu in a syngeneic, disseminated murine myeloid leukemia (B6SJLF1/J) model. Biodistribution studies showed that both ⁹⁰Y- and ¹⁷⁷Lu-anti-murine CD45 Ab conjugates (DOTA-30F11) targeted hematologic tissues, as at 24 hours 48.8±21.2 and 156±14.6% injected dose per gram of tissue (% ID/g) of ⁹⁰Y-DOTA-30F11 and 54.2±9.5 and 199±11.7% ID/g of ¹⁷⁷Lu-DOTA-30F11 accumulated in bone marrow (BM) and spleen, respectively. However, ⁹⁰Y-DOTA-30F11 RIT demonstrated a dose-dependent survival benefit: 60% of mice treated with 300 µCi ⁹⁰Y-DOTA-30F11 lived over 180 days after therapy, and mice treated with 100 µCi ⁹⁰Y-DOTA-30F11 had a median survival 66 days. ⁹⁰Y-anti-CD45 RIT was associated with transient, mild myelotoxicity without hepatic or renal toxicity. Conversely, ¹⁷⁷Lu- anti-CD45 RIT yielded no long-term survivors. Thus, ⁹⁰Y was more effective than ¹⁷⁷Lu for anti-CD45 RIT of AML in this murine leukemia model.     

Mycobacterium abscessus Morphotype Comparison in a Murine Model

Pulmonary infections with Mycobacterium abscessus (M. abscessus) are increasingly prevalent in patients with lung diseases such as cystic fibrosis. M. abscessus exists in two morphotypes, smooth and rough, but the impact of morphotype on virulence is unclear. We developed an immune competent mouse model of pulmonary M. abscessus infection and tested the differences in host inflammatory response between the morphotypes of M. abscessus. Smooth and rough morphotypes of M. abscessus were isolated from the same American Type Culture Collection strain. Wild type and cystic fibrosis mice were intratracheally inoculated with known quantities of M. abscessus suspended in fibrin plugs. At the time of sacrifice lung and splenic tissues and bronchoalveolar lavage fluid were collected and cultured. Bronchoalveolar lavage fluid was analyzed for leukocyte count, differential and cytokine expression. Pulmonary infection with M. abscessus was present at both 3 days and 14 days post-inoculation in all groups at greater levels than systemic infection. Inoculation with M. abscessus rough morphotype resulted in more bronchoalveolar lavage fluid neutrophils compared to smooth morphotype at 14 days post-inoculation in both wild type (p = 0.01) and cystic fibrosis (p<0.01) mice. Spontaneous in vivo conversion from smooth to rough morphotype occurred in 12/57 (21%) of mice. These mice trended towards greater weight loss than mice in which morphotype conversion did not occur. In the described fibrin plug model of M. abscessus infection, pulmonary infection with minimal systemic dissemination is achieved with both smooth and rough morphotypes. In this model M. abscessus rough morphotype causes a greater host inflammatory response than the smooth based on bronchoalveolar lavage fluid neutrophil levels.     

Evaluation of Tempol Radioprotection in a Murine Tumor Model

Tempol, a stable nitroxide free radical compound, is an in vitro and in vivo radioprotector. Previous studies have shown that Tempol protects C3H mice against whole-body radiation-induced bone marrow failure. In this study, the radioprotection of tumor tissue was evaluated. RIF-1 tumor cells were implanted in female C3H mice 10 d prior to radiation. Groups of mice were injected intraperitoneally with Tempol (275 mg/kg) or PBS followed 10 min later by a single dose of radiation to the tumor bed. Tumor growth curves generated after 10 and 33.3 Gy doses of radiation showed no difference in growth between the Tempol- and PBS-treated animals. A full radiation dose-response experiment revealed a tumor control dose in 50% of the animals in 30 d (TCD_50/30) value of 36.7 Gy for Tempol-treated mice and 41.8 Gy for saline-treated mice suggesting no protection of the RIF-1 tumor by Tempol. Tumor pharmacokinetics were done to determine why Tempol differentially protected bone marrow and not tumor cells. Differential reduction of Tempol in the RIF-1 tumor and bone marrow was evaluated with EPR spectroscopy 10, 20, and 30 min after injection. Bioreduction of Tempol to its corresponding hydroxylamine (which is not a radioprotector) occurred to a greater extent in RIF-1 tumor cells compared to bone marrow. We conclude that the differences in radioprotection may result from enhanced intratumor bioreduction of Tempol to its nonradioprotective hydroxylamine analogue. The nitroxides as a class of compounds may provide a means to exploit the redox differences between normal tissues and tumors. © 1997 Elsevier Science Inc.     

Murine Model of Hindlimb Ischemia

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate.In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.¹.     

Bactofection with Toll-Like Receptor 4 in a Murine Model of Urinary Tract Infection

The role of innate immunity in the prevention of urinary tract infection is well-documented. Toll-like receptor 4 (TLR4) is a major determinant of innate immune response. In an animal model of urinary tract infection, bactofection-mediated gene transfer of TLR4 was tested in a preventive approach. Bactofection with TLR4 reduced the colonization with uropathogenic Escherichia coli by 91% in the kidney and by 41% in the bladder. Reduced colonization was associated with lower oxidative stress and expression of monocyte chemoattractant protein-1 and myeloperoxidase in the kidney. Bactofection with TLR4 was successful in the prevention of ascending pyelonephritis. Further studies should focus on long-term effects, the dose response and the potential therapeutic use in models of chronic urinary tract infection.     

可移植性小鼠组织细胞肉瘤侵袭与转移模型的建立

将小鼠可移植性组织细胞肉瘤ＬⅡ分别接种在近交系６１５小鼠的胁部皮下、后肢肌肉和爪垫皮下组织内,取全肺和各部位淋巴结进行组织学检查,观察肿瘤自发转移率及转移程度。胁部皮下与后肢肌肉移植组待荷瘤小鼠自然死亡,平均存活时间分别为３９．３、２７．９ｄ,淋巴结转移率分别为９５．７％和１００％,肺转移率均为１００％。爪垫皮下移植组在肿瘤接种后１、３、５、１０、２０、３０和４０ｄ处死荷瘤小鼠,早期淋巴结转移出现在肿瘤接种后第１０天,至第３０天时形成肺转移瘤。局部侵袭达Ⅲ～Ⅳ级时才发生肿瘤转移,肺转移出现时间晚于淋巴转移。结果表明,ＬⅡ瘤株具有淋巴道合并血道转移的特性,是研究肿瘤侵袭和转移的理想实验肿瘤模型。     

Establishment of a Novel Murine Model of Ischemic Cardiomyopathy with Multiple Diffuse Coronary Lesions

Objectives

Atherosclerotic lesions of the coronary arteries are the pathological basis for myocardial infarction and ischemic cardiomyopathy. Progression of heart failure after myocardial infarction is associated with cardiac remodeling, which has been studied by means of coronary ligation in mice. However, this ligation model requires excellent techniques. Recently, a new murine model, HypoE mouse was reported to exhibit atherogenic Paigen diet-induced coronary atherosclerosis and myocardial infarction; however, the HypoE mice died too early to make possible investigation of cardiac remodeling. Therefore, we aimed to modify the HypoE mouse model to establish a novel model for ischemic cardiomyopathy caused by atherosclerotic lesions, which the ligation model does not exhibit.

Methods and Results

In our study, the sustained Paigen diet for the HypoE mice was shortened to 7 or 10 days, allowing the mice to survive longer. The 7-day Paigen diet intervention starting when the mice were 8 weeks old was adequate to permit the mice to survive myocardial infarction. Our murine model, called the “modified HypoE mouse”, was maintained until 8 weeks, with a median survival period of 36 days, after the dietary intervention (male, n = 222). Echocardiography demonstrated that the fractional shortening 2 weeks after the Paigen diet (n = 14) significantly decreased compared with that just before the Paigen diet (n = 6) (31.4±11.9% vs. 54.4±2.6%, respectively, P<0.01). Coronary angiography revealed multiple diffuse lesions. Cardiac remodeling and fibrosis were identified by serial analyses of cardiac morphological features and mRNA expression levels in tissue factors such as MMP-2, MMP-9, TIMP-1, collagen-1, and TGF-β.

Conclusion

Modified HypoE mice are a suitable model for ischemic cardiomyopathy with multiple diffuse lesions and may be considered as a novel and convenient model for investigations of cardiac remodeling on a highly atherogenic background.     

MCMV感染同种异型皮肤移植小鼠结肠炎模型的建立

利用小鼠巨细胞病毒(Murine cytomegalovirus,MCMV)感染同种异型皮肤移植小鼠,建立MCMV感染结肠炎症模型,从而为研究人类肠道疾病提供可靠的动物模型。采用鼻腔接种的方式感染同种异型皮肤移植的小鼠。①供体:C57BL/6雌鼠,18只;受体:BALB/c雌鼠,72只,鼠龄4～6周,体重14～20g/只;将C57BL/6小鼠的背部皮肤移植到BALB/c小鼠背部的移植床上,术后给BALB/c小鼠腹腔注射环孢素连续2周(12mg/kg.d)。②将移植后小鼠随机分组,每组24只,按接种病毒接种剂量分为104PFU组和105PFU组,同时设立阴性对照组,即鼻腔接种细胞悬液(1×106/mL)。每日观察动物排便情况及体重等总体情况变化;分别于病毒接种后第5、9、14和第21d取得小鼠结肠组织,通过组织病理学检测、原位杂交g、B RT-PCR、pp65免疫组化以及透射电镜的方法,观察检测小鼠结肠组织中MCMV与其组织病理变化之间的关联性。结果:在105PFU组中,小鼠出现厌食、嗜睡、活动能力明显下降,且发现该组小鼠的体重下降。在本研究中我们检验感染后第14d小鼠的结肠组织,发现感染组小鼠的近端结肠组织中粘膜层均变薄,同时其结构也被破坏;感染组小鼠的远端结肠组织中均出现淋巴样滤泡和粘膜层结构异常,其中105PFU组小鼠的结肠粘膜层的破坏得更严重;pp65免疫组化检验结果为MCMV蛋白阳性;原位杂交结果显示结肠组织中MCMV IE1基因阳性,MCMV gB基因RT PCR检测结果为阳性;透射电镜观察可见疱疹样病毒颗粒。而阴性对照组上述检测指标均为阴性。结果表明,在同种异型皮肤移植后小鼠鼻腔接种MC-MV,小鼠结肠发生了类似于人类结肠炎的病理变化。该结肠炎动物模型的建立将为进一步研究HCMV感染结肠的发病机理以及药物干预建立一个极为重要的平台。     

Nanocomposite Treatment Reduces Disease and Lethality in a Murine Model of Acute Graft-versus-Host Disease and Preserves Anti-Tumor Effects

Graft versus host disease (GVHD) is an immunological disorder triggered by bone marrow transplantation that affects several organs, including the gastrointestinal tract and liver. Fullerenes and their soluble forms, fullerols, are nanocomposites with a closed symmetrical structure with anti-inflammatory and anti-oxidant properties. The present study evaluated the effects of treatment with the fullerol (C60(OH)18-20) in the development and pathogenesis of GVHD in a murine model. Mice with experimental GVHD that were treated with the fullerol showed reduced clinical signs of disease and mortality compared with untreated mice. Treatment with the fullerol decreased the hepatic damage associated with reduced hepatic levels of reactive oxygen species, pro-inflammatory cytokines and chemokines (IFN-γ TNF-α, CCL2, CCL3 and CCL5) and reduced leukocyte accumulation. The amelioration of GVHD after treatment with the fullerol was also associated with reduced intestinal lesions and consequent bacterial translocation to the blood, liver and peritoneal cavity. Moreover, the fullerol treatment alleviated the GVHD while preserving effects of the graft against a leukemia cell line (GFP+P815). In summary, the fullerol was effective in reducing the GVHD inflammatory response in mice and may suggest novel ways to treat this disease.