Expression of a type 2 pneumocyte-specific antigen by a cell strain from normal adult mouse lung

The mouse lung epithelial cell strain NAL 1A has been confirmed as type 2 pneumocyte-related by immunostaining with a type 2 pneumocyte-specific antiserum. Cytoplasmic immunoreactivity with the antiserum paralleled the appearance of phospholipid-containing osmiophilic cytoplasmic inclusions, which were much more abundant in confluent cell cultures at low passage number than in exponentially growing cultures. However, phospholipid analysis indicated that NAL 1A cells were impoverished in phosphatidylglycerol as compared to lung type 2 pneumocytes. Confluent cultures of the neoplastic cell lines NAL 1AM and NUL 1 did not reveal any reactivity with the specific antiserum.     

Establishment of epithelial cell strains from normal adult mouse lung resembling a urethane-induced lung adenoma cell strain and a metastasizing mouse lung carcinoma cell line

Epithelial cell strains, designated NAL, have been established from normal lungs of adult female Balb/c mice. The ultrastructural characteristics, effect of dexamethasone on cellular morphology and proliferation rate, and the cytoskeletal protein pattern of NAL suggests that these cell strains may be related to a urethane-induced mouse lung adenoma cell strain NUL and to a metastasizing mouse lung tumour cell line CMT. NAL exhibited no anchorage-independent growth under normal conditions, however extensive passaging in the presence of 5 X 10(-6)M dexamethasone resulted in a colony forming efficiency in soft agar of 8.4% at passage number 30.     

Cloned murine non-malignant, spontaneously transformed and chemical tumour-derived cell lines related to the type 2 pneumocyte

NAL1A is a murine type 2 pneumocyte-related cell line cultured from normal BALB/c adult mouse lung. In vitro spontaneous transformation of 3 out of 7 clones of NAL1A has led to the isolation and establishment in continuous cell culture of sibling-related non-neoplastic (NAL1A) and spontaneously arising neoplastic (NAL1As) cell strains. NAL1As cells exhibited a similar phenotype to cloned NUL1 cells cultured from urethane-induced mouse lung adenomas. All NAL1As and NUL1 clones grew vigorously in 0.3% agar and formed invasive, poorly differentiated carcinomas following subcutaneous inoculation into immunesuppressed mice. Several subcutaneous nodules metastasised preferentially to the lung. All spontaneous and chemically-derived malignant clones were less differentiated than the non-malignant clones as assessed by staining with a type 2 pneumocyte-specific polyclonal antiserum. The clones described in this report form a useful model in the study of spontaneous and chemically-induced neoplastic transformation in mouse epithelial lung cells.     

季节性流感病毒 H1N1 BALB / c 鼠肺适应株的建立及其分子机制简

目的 建立季节性流感病毒H1N1的鼠肺适应株,并对适应的分子机理进行研究.方法 以病毒滴鼻感染小鼠,通过在BALB/c小鼠肺组织中连续传代,观察小鼠存活情况及肺病理改变,来获得季节性流感病毒H1N1的鼠肺适应株.结果季节性流感H1N1 A/Brisbane/59/2007病毒野生型毒株,经过在小鼠体内进行8次传代后,毒力逐渐增强,从无致病力到致死率达到100%,对鼠肺适应株与野生型毒株进行基因比对,发现适应株HA基因发生了3个有义突变.结论 野生季节性低致病力H1N1流感病毒可经在小鼠中经过多次传代而获得高致病力H1N1鼠肺适应株,HA蛋白89位Thr至Ile的突变对毒力的增强起决定性作用.     

狂犬病病毒CTN-1V株在人二倍体细胞Walvax-2株的适应传代

目的建立狂犬病病毒固定毒CTN-1V株在人二倍体细胞Walvax-2株上的传代适应株。方法用狂犬病病毒固定毒CTN-1V株经昆明小鼠鼠脑回传后的病毒接种Walvax-2细胞,连续传代,检测各代次病毒的滴度及免疫原性。结果 CTN-1V株能较好地适应Walvax-2细胞,通过连续带毒传代至第7代,病毒滴度可达6.78 lg LD50/mL,并在第10~15代内滴度维持在7.0 lg LD50/mL以上,15~30代滴度稳定在7.0 lg LD50/mL左右。以15代适应毒株CTN-1V-HDC P15制备的疫苗原液,各项指标均符合《中华人民共和国药典》三部(2010版)的要求,疫苗效力在6.0IU/剂以上。结论所获人胚肺二倍体细胞Walvax-2株传代适应狂犬病毒固定毒株CTN-1V-HDC可用于人用狂犬病疫苗的生产开发。     

Radiation-induced lung response of AcB/BcA recombinant congenic mice

Lemay, A-M. and Haston, C. K. Radiation-Induced Lung Response of AcB/BcA Recombinant Congenic Mice. Radiat. Res. 170, 299-306 (2008).The genetic factors that influence the development of radiotherapy-induced lung disease are largely unknown. Herein we identified a strain difference in lung response to radiation wherein A/J mice developed alveolitis with increased levels of pulmonary mast cells and cells in bronchoalveolar lavage while the phenotype in C57BL/6J mice was fibrosis with fewer inflammatory cells. To identify genomic loci that may influence these phenotypes, we assessed recombinant congenic (RC) mice derived from the A/J and C57BL/6J strains for their propensity to develop alveolitis or fibrosis after 18 Gy whole-thorax irradiation. Mouse survival, lung histopathology and bronchoalveolar lavage cell types were recorded. Informative strains for each of mast cell influx, bronchoalveolar cell numbers, alveolitis and fibrosis were identified. In mice with the A/J strain background, the severity of alveolitis correlated with increased mast cell numbers while in C57BL/6J background strain mice fibrosis was correlated with the percentage of neutrophils in lavage. The data for RC mice support the association of specific inflammatory cells with the development of radiation-induced lung disease and provide informative strains with which to dissect the genetic basis of these complex traits.     

恶性横纹肌样瘤（MRT）的起源研究——高变异率HeLa细胞致裸鼠产生MRT的实验研究

HeLa细胞KB株、X株、NM20／X株、H株的染色体众数依次为60±3（超二倍体）、62±3（超二倍体）、68±3（超二倍体和亚四倍体）和78±2（亚四倍体）,所占比率分别为72%～76%,69%,52%和40%。在纯化3代的肿瘤阴性对照二倍体猫肾（染色体众数38所占比率80%）和犬肾原代细胞皮下接种裸鼠的致癌／致瘤率分别为0%（0／22）和0%（0／10）,X株HeLa细胞冻融裂解物皮下接种裸鼠产生进行性缩小肿瘤的比率为20%（1／5）的前提下,HeLa细胞KB株、X株、NM20／X株皮下接种裸鼠产生进行性生长恶性肿瘤的比率分别为100%（10／ 10）,100%（25／25）和100%（5／5）,H株细胞皮下接种裸鼠产生恶性肿瘤的比率为50%（5／10）。其中,只有HeLa细胞KB株10～11代（染色体结构畸变率高达20%,出现18%双着丝点和2%断片）以超高数量接种的1组4只裸鼠（0.17ml12.75×10     

病毒疫苗制备用不同核型VERO细胞系在裸鼠体内致癌/致瘤性的系统实验研究

生产疫苗用细胞系可能具有致瘤性,一些常用的细胞系需要检查不同代次有无致癌性。在建立传代细胞种子库与工作库基础上,对研制生产病毒活疫苗所用8株VERO细胞系在219只裸鼠进行了致癌(瘤)实验。本研究结果表明,VERO细胞染色体核型可发生变异,亚四倍体JA株与超二倍体KA株具有强的致癌性,不能用于致弱活病毒疫苗制备,但可替代HeLa细胞系用作恶性肿瘤阳性对照细胞。筛出无致瘤性的YB、dC、M和JB株亚二倍体VERO细胞系,可替代BHK-21细胞用于狂犬病减毒活疫苗制备。VERO细胞系染色体遗传相对稳定。不同代次变化不大。研究发现细胞染色体遗传特征决定致瘤性质并具有种属特异性,不同核型细胞致瘤性不同,细胞染色体数目变异大小和致癌性成正相关,通过体内外交替选育可在裸鼠体内快速选育成功高变异率肿瘤细胞系。高变异率HeLa或VERO细胞系移植于裸鼠可能产生恶性横纹肌样瘤。因此,应当强调疫苗生产用细胞系致瘤性评价的重要性。     

KLN205—A murine lung carcinoma cell line

Summary KLN205 cells, a cloned cell line established from the Nettesheim lung carcinoma, grow in various synthetic media such as MEM, Fisher's or Roswell Park Memorial Institute Medium (RPMI) with the addition of 5 to 20% fetal bovine serum (FBS), calf serum (CS) or horse serum (HS). They grow optimally in minimum Eagle's medium plus nonessential amino acids (NEAA) plus 5 to 10% FBS or HS. The cells are transplantable to DBA/2, BDF₁, AKD₂F₁, and BALB/c, but not to C3H/He or ICR mice. The growth curves, plating efficiency, ultrastructural characteristics, modal number of chromosomes and transplantability to mice of various strains are almost the same for early and late passages of cells passaged in vitro. These parameters for 16th and 36th passages were: doubling time, 31 and 33 hr; plating efficiency, 12.4±1.2 and 14.6±2.6%; modal number of chromosomes, 73 and 76; lung colony formation in DBA/2, 50 and 45.9/mouse; and subcutaneous tumor diameter 24.5 and 27.4 mm, respectively. Only the numbers of lung colonies formed in BDF₁ mice were different: 24.4/mouse with 16th passage cells, and 10.2/mouse with 36th passage cells. The results suggest that KLN205 is a relatively stable cultured cell line through 36 passages. As was expected, immunosuppression by higher concentrations of triaminolone acetonide (TA) enhanced lung colony formation in BDF₁ mice. On the other hand, a low concentration of TA inhibited lung colony formation in DBA/2 mice, which was unexpected. These results suggest that KLN205 offers a model for investigations on metastases to lungs as well as chemotherapy for lung carcinoma. This work was supported by the National Cancer Institute of Canada.     

Bovine Epizootic Fever

A virus, the Yamaguchi strain, was serially propagated in suckling hamsters, mice and rats, and hamster kidney BHK21-WI2 cells from a natural case in the 1966 outbreak of bovine epizootic fever, an acute febrile disease of cattle, resembling ephemeral fever, known in Japan since 1949. An acute phase blood from the natural case was first passaged in calves by intravenous inoculation, and a blood specimen at the second passage was used to initiate serial hamster passage. Infected hamsters died with nervous symptoms, and serial passage was readily accomplished by intracerebral inoculation with brain emulsions. The hamster passage line of virus was serially passaged by intracerebral inoculation in 1- or 2-day-old mice, and then in rats 1 or 2 days after birth, which developed fatal encephalitis. The hamster passage virus was also serially propagated with cytopathic effect in cultures of BHK21-WI2 cell cultures. These viral lines were shown to be neutralized by, and to produce specific complement-fixing antigen reactive with, convalescent sera of calves infected with the original Yamaguchi strain, confirming the identity of these lines as the Yamaguchi strain. The hamster passage line, when inoculated intravenously in calves, induced an acute febrile illness which was similar to bovine epizootic fever; all the inoculated calves had viremia and developed neutralizing and complement-fixing antibodies against the virus. Serological evidence for infection with this virus was obtained in natural cases in the 1966 outbreak. These findings seem to justify this virus to be the causative agent of bovine epizootic fever.     

Role of rpoS in acid resistance and fecal shedding of Escherichia coli O157:H7

Acid resistance (AR) is important to survival of Escherichia coli O157:H7 in acidic foods and may play a role during passage through the bovine host. In this study, we examined the role in AR of the rpoS-encoded global stress response regulator sigma(S) and its effect on shedding of E. coli O157:H7 in mice and calves. When assayed for each of the three AR systems identified in E. coli, an rpoS mutant (rpoS::pRR10) of E. coli O157:H7 lacked the glucose-repressed system and possessed reduced levels of both the arginine- and glutamate-dependent AR systems. After administration of the rpoS mutant and the wild-type strain (ATCC 43895) to ICR mice at doses ranging from 10(1) to 10(4) CFU, we found the wild-type strain in feces of mice given lower doses (10(2) versus 10(3) CFU) and at a greater frequency (80% versus 13%) than the mutant strain. The reduction in passage of the rpoS mutant was due to decreased AR, as administration of the mutant in 0.05 M phosphate buffer facilitated passage and increased the frequency of recovery in feces from 27 to 67% at a dose of 10(4) CFU. Enumeration of E. coli O157:H7 in feces from calves inoculated with an equal mixture of the wild-type strain and the rpoS mutant demonstrated shedding of the mutant to be 10- to 100-fold lower than wild-type numbers. This difference in shedding between the wild-type strain and the rpoS mutant was statistically significant (P 相似文献   

Activation of metastatic potential in african green monkey kidney cell lines by prolongedin vitro culture

Summary Studies on the tumorigenicity of Vero kidney cells ofCercopithecus aethiops monkey origin were extended to various passage levels of BSC-1 aneuploid cells and to low passage CV-1 diploid cells (derived also fromC. aethiops monkey kidney). It was found that BSC-1 cells—like Vero cells—showed increased tumorigenicity with increasing passage level in antitymocyte globulins (ATG) treated newborn rats and in nude mice. Cells passaged over 250 times in cultures formed invasive adenocarcinomas in newborn rats. Their malignant tumor growth was further demonstrated around the 500 passage level when tumor metastases were detected in the lungs of four of the 14 inoculated rats. Vero cells induced such lung metastases in rats already at passage 227. CV-1 diploid cells at low passage level produced small nodules of epithelioid cells in newborn rats at 6th day after inoculation that had disappeared by the 21st day, and caused no local invasion nor lung metastasis. In vitro tumorigenicity tests on BSC-1 and CV-1 cells, using chick embryo skin, human muscle and colony formation in agarose, confirmed the animal test results. The results of this study indicate that BSC-1 and Vero cell lines at low and high passage levels may prove to be useful tools to study the molecular basis of malignancy. Editor's Statement In this paper Contreras et al. document the increased malignancy of commonly-used monkey cell lines upon long-term culture. These observations have implications for the use of these cell lines in studies of cancer cell biology, as well as the use of these lines for the production of biologicals. David W. Barnes     

Immunohistological characterization of leukocytes in the lungs of healthy mice and after bacterial intratracheal infection

Leukocytes in the peripheral lung parenchyma of mice have not been characterized histologically during bacterial infection. The aim of this study was to investigate (a) the immunohistological characteristics of healthy murine lungs and (b) the cell kinetics during acute inflammation. BALB/c and MF1 mice were examined; as well as transgenic mice with the gene defect of cystic fibrosis (CF) in the airways as an animal model for this disease. MF1 mice served as controls for the transgenic animals. Lavaged and perfused lungs were snap frozen. B and T lymphocytes, CD4+ and CD8+ cells, dendritic cells, neutrophils and a subset of macrophages were enumerated on cryostat lung sections. The lung tissue and bronchoalveolar lavage (BAL) of BALB/c mice, infected intratracheally with Haemophilus influenzae type b (Hib), were studied at different time points after infection. In the lungs of healthy mice, including CF mice, the largest population was that of T cells, CD4+ cells being always more frequent than CD8+ cells. During acute inflammation the number of neutrophils in the lung parenchyma and BAL increased strongly within the first hours after bacterial instillation and reached baseline levels within one week. This study provides a semi-quantitative analysis of immunocompetent cells in normal and infected murine lung tissue. Differences in cell numbers are found between different strains. Moreover, the cellular reaction during Hib infection in mouse lungs is dominated by neutrophils, as expected in a primary immune response. In uninfected CF mice the numbers and distribution of immune cells in the lung tissue are normal, indicating that the cellular defense is adequate.     

An attenuated strain of Akabane virus: a candidate for live virus vaccine

An attempt was made to attenuate the high virulent OBE-1 strain of Akabane virus by adaptation to low temperature. In it the virus was subjected to passage through HmLu-1 cell cultures at 30 degrees C. Cloning was carried out on the virus which had undergone 20 passages through these cultures to select a strain adapted to low temperature. Finally, ten clones were obtained. As a result, nine strains of clone in which virus replication was poor in HmLu-1 cell cultures at 40 degrees C were obtained. Of them, five strains of clone produced uniform plaques. Of these strains, one, or the TS-C2 strain, was selected. It was considerably lower both in peripheral infectivity to suckling mice and in intracerebral infectivity to 3-week-old mice than the OBE-1 strain. Calves and pregnant cows inoculated with the TS-C2 strain by the intracerebral, intravenous, or subcutaneous route were free from pyrexia, leukopenia, and viremia. Virus recovery was negative from various organs and fetuses. All the animals inoculated, however, were found to have neutralizing antibody produced. The results mentioned above suggested that the TS-C2 strain might have been so attenuated as to be available as a candidate strain for a live virus vaccine.     

Characteristic faecal flora of NC mice

The composition of faecal flora of NC mice was compared with that of CF #1 mice. NC- and CF #1-germfree (GF) mice were cage-mated with NC- or CF #1-conventional (CV) mice in an isolator. The faecal flora of these ex-GF mice was dependent on the recipient mouse strain modifying colonization by the donor mouse bacteria. Although NC- and CF #1-pups removed by hysterectomy were fostered to different strains, almost all these mice at 8 weeks old had a strain characteristic pattern of faecal flora regardless of the foster strains. In GF mice mono-associated with a Lactobacillus strain or a Bifidobacterium strain isolated from faeces of CV mice, the numbers of these bacteria in the stomach and small intestine of NC mice were lower than those of CF #1 mice. In GF mice associated with chloroform-treated faeces of CV mice, and a Lactobacillus strain or a Bifidobacterium strain, the numbers of these bacteria in the stomach and all parts of the intestine of NC mice were considerably lower than those of CF #1 mice. These results suggested that the composition of faecal flora of NC mice were characteristic, i.e. the fact that the numbers of lactobacilli were low compared with CF #1 mice with ordinary faecal flora and the colonization of bifidobacteria, peptococcaceae and eubacteria on ES agar in NC mice intestine differed, was due to genetic factors.     

甲型肝炎重组痘苗病毒(VMS11HAV25)的构建及其某些生物学性质

将5′端经过剪切的甲型肝炎病毒全部开放读码框架cDNA连接于痘苗病毒晚期启动子P11下游,重组于痘苗病毒天坛株的HiodⅢM片段Spb Ⅰ位点获得了重组病毒VMS11HAV25。对其生物学性质的研究表明,该重组病毒诱生痘苗抗体的能力、对鸡红细胞的血凝性质、空斑大小及对温度的稳定性等均与原天坛株相同。重要的区别是,重组病毒在家兔皮内和小鼠脑内的毒力都比原天坛株低约1个对数。病毒在人胚肺二倍体细胞连续传15代的表达水平与传代早期者相同。连续传20代后提取病毒DNA做Southern blot杂交表明,甲型肝炎病毒基因仍稳定地存在于原插入位置。     

Th1-Th2 cytokine kinetics in the bronchoalveolar lavage fluid of mice infected with Cryptococcus neoformans of different virulences

Th1 immune response plays an important role in protection against infection with Cryptococcus neoformans in mice. We investigated the effect of virulence of C. neoformans on cytokine production in the lung of a mouse model of pulmonary cryptococcosis. BALB/c mice were inoculated intratracheally with a high or low virulence strain of C. neoformans, followed by serial measurements of Th1 and Th2 cytokine concentrations in the bronchoalveolar lavage (BAL) fluid using appropriate enzyme-linked immunosorbent assay kits. The number of colony-forming units (CFU) increased with time, and all mice infected with the highly virulent strain were dead at 28 days after inoculation. In contrast, the number of microorganisms diminished with time in the mice infected with the low virulence strain during the 4-week study. The numbers of neutrophils and lymphocytes in the BAL fluid paralleled those of CFU. High neutrophil counts were observed in the BAL fluid of mice infected with the highly virulent strain, while lymphocyte counts were increased only in the later part of the study in mice infected with the high and low virulence strains. The concentrations of Th2 cytokine, interleukin (IL)-4 were significantly higher in mice infected with the highly virulent strain at days 14 and 21 of infection, whereas the level of Th1 cytokine, interferon-gamma, was significantly higher in the latter strain at days 7 and 14. Our results suggest that strain-specific difference in the organism's ability to induce (or evade) the host immune system contributes to the outcome of infection.     

Placenta as a selective barrier to cellular traffic

Transplacental passage of cells from mother to fetus during murine pregnancy was examined by using glucose phosphate isomerase (GPI) or fluorescein tagging as markers. Female mice of strains (BALB/cCr X C3H/HeJ)/F1 or (A/J X C57BL/6J)F1 (both Gpi-1a/b) were mated to the corresponding Gpi-la males (BALB/cCr or A/J, respectively). Cells of the liver, blood, and/or spleen in the offspring were typed at days 15, 16, 17, or 18 of gestation, the day of delivery, or 1 day postpartum. Only two of 172 Gpi-1a/a mice obtained from these matings showed evidence of maternal cell trafficking. Sensitivity of the assay was 1% Gpi-1a/a population. Fluorescein-labeled BALB/cCr peripheral red blood cells (RBC) or white blood cells (WBC) were injected i.v. into syngeneically mated BALB/cCr mothers on day 18. After 24 hr, the blood or liver of the neonates was formalin-fixed and examined in the fluorescence-activated cell sorter (FACS). Some RBC crossed the placenta, but WBC were usually not detected in fetal liver in significant numbers. This technique was very sensitive, and we estimated that no more than 225 WBC could enter the fetus via this route. Thus, we conclude that passage of significant numbers of maternal WBC into the fetus is rare, and perhaps this passage is related to a placental abnormality.