Comparison of methods to diagnose an epizootic of Corynebacterium kutscheri pneumonia in rats

An outbreak of Corynebacterium kutscheri pneumonia occurred in a colony of laboratory rats. Diagnostic methods compared on a prospective basis included (1) an enzyme-linked immunosorbent assay (ELISA) for serum antibody to C. kutscheri; (2) C. kutscheri isolation from retrograde nasal wash, cervical lymph nodes, tracheal wash, lung homogenate; and (3) histopathology. C. kutscheri was isolated from one or more of the cultured sites from suspected cases, but no individual rat yielded C. kutscheri from all of the sites. The presence of histological lung lesions was the most frequent indicator of infection (8 of 9 suspected cases), followed by isolation of C. kutscheri from lung homogenate (5 of 6), ELISA for serum antibody (6 of 8), and isolation of C. kutscheri from cervical lymph node (4 of 8). Isolation of C. kutscheri from nasal wash (1 of 9) was the least sensitive indicator of infection. ELISA antibody was not detected in rats which had normal lungs and did not harbor C. kutscheri. It was concluded that ELISA provides noninvasive means for detecting infection and cervical lymph node culture increase the potential for successful isolation of the agent in C. kutscheri infected rats.     

Role of galectin-3 as an adhesion molecule for neutrophil extravasation during streptococcal pneumonia

Recruitment of neutrophils from blood vessels to sites of infection represents one of the most important elements of innate immunity. Movement of neutrophils across blood vessel walls to the site of infection first requires that the migrating cells firmly attach to the endothelial wall. Generally, neutrophil extravasation is mediated at least in part by two classes of adhesion molecules, beta(2) integrins and selectins. However, in the case of streptococcal pneumonia, recent studies have revealed that a significant proportion of neutrophil diapedesis is not mediated by the beta(2) integrin/selectin paradigm. Galectin-3 is a beta-galactoside-binding lectin implicated in inflammatory responses as well as in cell adhesion. Using an in vivo streptococcal pneumonia mouse model, we found that accumulation of galectin-3 in the alveolar space of streptococcus-infected lungs correlates closely with the onset of neutrophil extravasation. Furthermore, immunohistological analysis of infected lung tissue revealed the presence of galectin-3 in the lung tissue areas composed of epithelial and endothelial cell layers as well as of interstitial spaces. In vitro, galectin-3 was able to promote neutrophil adhesion to endothelial cells. Promotion of neutrophil adhesion by galectin-3 appeared to result from direct cross-linking of neutrophils to the endothelium and was dependent on galectin-3 oligomerization. Together, these results suggest that galectin-3 acts as an adhesion molecule that can mediate neutrophil adhesion to endothelial cells. However, accumulation of galectin-3 in lung was not observed during neutrophil emigration into alveoli induced by Escherichia coli infection, where the majority of neutrophil emigration is known to be beta(2) integrin dependent. Thus, based on our results, we propose that galectin-3 plays a role in beta(2) integrin-independent neutrophil extravasation, which occurs during alveolar infection with Streptococcus pneumoniae.     

The effect of selected viruses on Corynebacterium kutscheri infection in rats

The influence of selected viral pathogens on rats that were previously infected with Corynebacterium kutscheri was investigated. A series of three separate experiments were performed to test the effect of sialodacryoadenitis virus, Sendai virus and rat virus. In each experiment, weanling rats were divided into three groups (C. kutscheri-inoculated, virus-inoculated and C. kutscheri plus virus-inoculated). Two groups were inoculated oronasally with C. kutscheri to establish subclinical infections. Two weeks later, two groups were inoculated intranasally with virus. At 5 weeks, the prevalence of C. kutscheri recovery from oral cavity and submaxillary lymph node and the prevalence of overt pseudotuberculosis was compared between treatment groups. Seroconversion of rats to C. kutscheri was measured by microagglutination and viruses by indirect immunofluorescence assays. Infection of rats with sialodacryoadenitis virus, Sendai virus or rat virus had no discernable effect on C. kutscheri-infected rats.     

Serological surveys of Corynebacterium kutscheri infection in mice and rats

Serological surveys of mice and rats naturally infected with Corynebacterium kutscheri were performed by examining serum samples collected from breeder and laboratory colonies between 1981 and 1983. Among 756 mice from 73 conventional colonies, only 4 animals (0.5%) from 3 colonies (4.1%) developed C. kutscheri antibody of 1:40 to 1:2, 560 titers. Three of them suffered from abscess caused by the organism. Regarding a titer of 1:40 or higher as reliably positive, 87 (13.0%) of 669 conventional rats or 20 (32.8%) of 61 colonies were found to be infected with the organism. The antibodies were detected in both types of animals older than 6 months of age. No lesions caused by C. kutscheri were found in almost all the rats examined. Germ-free and SPF mice and rats were all negative for antibody at 1:5 serum dilution.     

Detection of Corynebacterium kutscheri from the oral cavity of rats.

A simple and useful method for the detection of C. kutscheri from the oral cavity of living rats was devised. In 10 sacrificed rats from two naturally and subclinically infected conventional colonies, 10(4.28) or 10(3.84) CFU/ml C. kutscheri were isolated from upper incisor swab extractions, while 10(1.38) or 10(1.58) and < 10 or 10(1.56) CFU/ml from the upper soft palate and pharynx, respectively. In another survey with 26 living animals, which were reared on the same rack, organisms were detected from the upper incisor and gingival swabs in 15 of 26 rats (57.7%). The results were reproducible at a second survey 10 days later. No organisms were isolated from any sites of the orally negative rats. These results indicated that culture of swab specimens from the upper incisors and gingivae of incisors is useful for the detection of C. kutscheri infection in living rats.     

Cell-type specific regulation of galectin-3 expression by glucocorticoids in lung Clara cells and macrophages

Bronchiolar Clara cells are integral components of lung homeostasis, predominantly distributed in distal airways. In addition to the 16 kDa Clara cell protein, a major secretory product with anti-inflammatory effects, rat Clara cells express the glycan-binding protein galectin-3 and secrete it into the airways. Given the essential role of galectin-3 in the control of inflammation and the well-established function of glucocorticoids (GCs) in lung physiology, here we investigated whether galectin-3 is a target of the regulatory effects of GCs. Adult male rats were subjected to bilateral adrenalectomy and the lungs were processed for light and transmission electron microscopy, immunoelectron microscopy and Western blot analysis. Profound changes in bronchiolar Clara cells and macrophage morphology could be observed by electron microscopy after adrenalectomy. While specific galectin-3 staining was detected in the nucleus and cytoplasm of Clara cells and macrophages from control animals, cytoplasmic galectin-3 expression was dramatically reduced after adrenalectomy in both cell types. This effect was cell-specific as it did not affect expression of this lectin in ciliated cells. After dexamethasone treatment, galectin-3 expression increased significantly in the nucleus and cytoplasm of macrophages and Clara cells. Western blot analysis showed a clear decrease in galectin-3 expression in ADX animals, which was recovered after a 7-day treatment with dexamethasone. In peritoneal macrophages, galectin-3 expression was also dependent on the effects of GCs both in vivo and in vitro. Our results identify a cell type-specific control of galectin-3 synthesis by GCs in lung bronchiolar Clara cells and interstitial macrophages, which may provide an alternative mechanism by which GCs contribute to modulate the inflammatory response.     

EPO对肾脏缺血再灌注损伤大鼠肺内氧化应激状态的影响

目的:探讨促红细胞生成素(erythropoietin,EPO)对大鼠肾脏再灌注损伤模型肺内氧化应激状态的影响。方法:清洁级Sprague-Dawly(SD)大鼠36只适应性喂养1周后,随机分为3组,即假手术组(A组)、肾脏再灌注损伤组(B组)和EPO预处理组(C组),每组12只。A组大鼠只打开腹腔,游离双侧肾蒂但不夹闭;B组与C组都建立了大鼠肾脏再灌注损伤模型,且C组在夹闭肾蒂前2 h腹腔注射人重组EPO(5000 U/kg)。术后24 h,处死大鼠,检测肺内氧化应激水平。结果:A组大鼠精神状态良好,肾小管结构正常,未见明显上皮细胞肿胀、脱落,肺泡结构基本完整,肺泡间隔未增厚,有少量炎性细胞浸润;B组鼠毛耸立,无光泽,饮水量减少,肾小管结构破坏消失,肾小管扩张,可见大量蛋白管型,肺泡结构破坏,肺泡腔缩窄,肺泡间隔增厚,组织水肿,大量炎性细胞浸润;C组大鼠精神状态有所恢复,一般状况尚可,肾小管损伤较B组轻似,肾小管坏死区域有所减少,坏死偶见,肺泡壁轻度破坏,结构较为清晰,可见少量炎性细胞浸润。B组与C组大鼠的血尿素氮(blood urea nitrogen, BUN)与血肌酐(serum creatinine,Scr)水平、肺组织血红素氧合酶(heme oxygenase, HO)-1与丙二醛(malondialdehyde, MDA)水平都显著高于A组,C组以上指标均显著低于B组(P0.05)。B组超氧化物歧化酶(superoxide dismutase, SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)的水平均显著高于A组(均P0.05),而C组SOD和GSH-Px的水平均显著高于B组(均P0.05),A组与C组间对比无显著差异。结论:EPO用于大鼠肾脏再灌注损伤模型能缓解肺内氧化应激状态,促进肾功能及肺组织恢复,发挥肾脏保护作用。     

Experimental Corynebacterium kutscheri infection in rats: bacteriology and serology

In a preliminary study, hydrocortisone-treated rats developed pseudotuberculosis when challenged with 6.2 X 10(5) to 3.1 X 10(7) colony forming units of Corynebacterium kutscheri by intranasal, intragastric, or subcutaneous inoculation. Oronasal exposure was selected as a likely natural route to further study inapparent infection. In Study 1, 50 rats received 1.2 X 10(5) colony forming units and various tissues were cultured at intervals to 12 weeks post-inoculation. At each interval, C. kutscheri was regularly isolated from submaxillary lymph nodes, but isolation was sporadic from other sites. In Study 2, 17 out of 21 rats given 1.2 X 10(5) colony forming units and killed weekly for 6 weeks had 2.0 X 10(2) to 1.8 X 10(5) colony forming units of C. kutscheri in oral washes, and 16 rats had 2.0 X 10(2) to 1.0 X 10(5) colony forming units in submaxillary lymph nodes, Serum antibody to C. kutscheri using both microagglutination and indirect immunofluorescence was first detected in some rats by 2 weeks, and in all rats at subsequent intervals. There was a significant (P less than 0.001) positive correlation (r = 0.93) between serum antibody titers and the duration of infection.     

Isolation of Corynebacterium kutscheri from aged Syrian hamsters (Mesocricetus auratus).

Corynebacterium kutscheri was isolated from the oral cavities of 12 male Syrian hamsters (Mesocricetus auratus) which were about 12 months old. At 1, 5, and 9 months after initial isolation of C. kutscheri from the oral cavity, hamsters were euthanatized, and attempts were made to culture C. kutscheri from 13 additional sites. Corynebacterium kutscheri was isolated from nine hamsters, and regardless of the hamsters' ages, the organisms were most frequently isolated from the oral cavity (100%), esophagus (100%), cecal content (100%), and colon and rectum (88.9%). Isolation rates in the nasal cavity were 66.7%, followed by 55.5% in the trachea and 33.3% in the submaxillary lymph nodes. The number of the organisms found in the submaxillary lymph nodes and esophagus was 10(3) to 10(4) CFU/g. The number found in the cecal content and in the colon and rectum was 10(2) to 10(5) CFU/g. The organisms were not isolated from lung, stomach, kidney, spleen, and mesenteric lymph node tissues. The hamsters had neither clinical signs nor lesions. However, 7 of 12 animals had low agglutinating antibody titers. The Syrian hamster can therefore be an asymptomatic carrier of C. kutscheri.     

The Axonal Guidance Cue Semaphorin 3C Contributes to Alveolar Growth and Repair

Lung diseases characterized by alveolar damage such as bronchopulmonary dysplasia (BPD) in premature infants and emphysema lack efficient treatments. Understanding the mechanisms contributing to normal and impaired alveolar growth and repair may identify new therapeutic targets for these lung diseases. Axonal guidance cues are molecules that guide the outgrowth of axons. Amongst these axonal guidance cues, members of the Semaphorin family, in particular Semaphorin 3C (Sema3C), contribute to early lung branching morphogenesis. The role of Sema3C during alveolar growth and repair is unknown. We hypothesized that Sema3C promotes alveolar development and repair. In vivo Sema3C knock down using intranasal siRNA during the postnatal stage of alveolar development in rats caused significant air space enlargement reminiscent of BPD. Sema3C knock down was associated with increased TLR3 expression and lung inflammatory cells influx. In a model of O₂-induced arrested alveolar growth in newborn rats mimicking BPD, air space enlargement was associated with decreased lung Sema3C mRNA expression. In vitro, Sema3C treatment preserved alveolar epithelial cell viability in hyperoxia and accelerated alveolar epithelial cell wound healing. Sema3C preserved lung microvascular endothelial cell vascular network formation in vitro under hyperoxic conditions. In vivo, Sema3C treatment of hyperoxic rats decreased lung neutrophil influx and preserved alveolar and lung vascular growth. Sema3C also preserved lung plexinA2 and Sema3C expression, alveolar epithelial cell proliferation and decreased lung apoptosis. In conclusion, the axonal guidance cue Sema3C promotes normal alveolar growth and may be worthwhile further investigating as a potential therapeutic target for lung repair.     

Pulmonary vascular reactivity in Fischer rats

We previously reported that Fischer (F) rat lungs developed more extensive injury when challenged with oxidants than age-matched Sprague-Dawley (SD) rat lungs. We now describe a reduced pulmonary vascular response to alveolar hypoxia and angiotensin II (ANG II) in F compared with SD rats. The comparative studies were performed with isolated lungs perfused with salt solution or blood, catheter-implanted awake rats, and isolated main pulmonary arterial rings. Isolated lungs from F rats perfused with either blood or salt solution had reduced vasoconstriction in comparison with lungs from SD rats when exposed to alveolar hypoxia or challenged with ANG II. Instrumented awake F rats had a smaller mean increase in total pulmonary vascular resistance (PVR) than SD rats (35 vs. 94 mmHg.min.l-1, P less than 0.05) when challenged with 8% oxygen. The contractile response of isolated pulmonary artery but not thoracic aortic rings to KCl and ANG II was reduced in F compared with SD rats. In addition, F rats exposed to 4 wk of hypobaric hypoxia developed less pulmonary hypertension and right ventricular hypertrophy (when corrected for the hematocrit) than SD rats. We conclude that the oxidant stress-sensitive inbred F rat strain is characterized by a lung vascular bed that is relatively unresponsive to vasoconstricting stimuli. The mechanism underlying this genetic difference in lung vascular control remains to be defined.     

Naturally occurring subclinical Corynebacterium kutscheri infection in laboratory rats: strain and age related antibody response

Naturally occurring subclinical Corynebacterium kutscheri infection was analyzed by antibody response related to the strain of rats. Wistar-Lewis, Wistar and Spraque-Dawley rats were high responders in seroconversion rates and antibody titers, while Brown Norway and Fischer rats were low responders. The antibody response was related to age also. Some young rats had maternal antibody to C. kutscheri, but antibody disappeared before 8 weeks of age. Rats were antibody-negative for several months thereafter and became antibody-positive after 6 months of age. The antibody response was highest at 8 to 9 months of age in subclinical C. kutscheri infection. This antibody response was very late, compared to the antibody response to Sendai virus and Mycoplasma infections.     

Alterations in gene expressions encoding preproET-1 and NOS in pulmonary tissue in endotoxemic rats

Septic shock is characterized by hypotension and a hyporeactive response to vasopressor agents. The pathogenesis is due to vascular leaks and an increased synthesis of cytokines and nitric oxide (NO). The present study examined the time-dependent alterations of endothelin-1 (ET-1) and the expression of NO synthase (NOS) in lung tissue in a septic rat model. Normal Sprague-Dawley (SD) rats aged 10 weeks received 15 mg/kg lipopolysaccharide (LPS) and then were sacrificed at different time points (1, 3, 6, and 10 hrs). Rats that did not receive LPS were considered to be controls. Both systolic and diastolic pressure decreased in SD rats after LPS administration. Time-dependent onset of features of acute lung injury, such as the infiltration of inflammatory cells and thickening of alveolar septa, were seen in rats that received LPS. A 2.8-fold increase in the expression of preproET-1 level was observed in lung tissue 6 hrs after LPS administration. The expression of endothelial NOS (eNOS) was also altered in lung tissue in a time-dependent fashion. After the administration of LPS, there was a 16-fold increase in the expression of eNOS mRNA. The peak expression of inducible NOS (iNOS) in lung tissue specimens obtained from rats that received LPS was 45-fold higher than that in control rats. ET-1 is a potent vasoconstrictor and thereby may play an important role in the pathogenesis of acute lung injury in a septic rat model. The increased expression of NOS may result in excess NO production and may also play a role in the pulmonary complications of endotoxemia.     

Pathological studies on corynebacterial ulcerative entero-colitis in rats treated with ACTH (author's transl)]

Ulcerative entero-colitis was developed in 6-week-old male Sprague-Dawley rats treated daily with ACTH (4 mg/kg. s. c.) as well as necrotic purulent lesions in liver, kidney, lung or heart. Incidence of ulcerative lesions was 6.3% in Farm-A rats and 56% in Farm-B rats. Although ulcerative lesions were mostly observed in cecum, the similar lesions were also detected in distal ileum or proximal colon in some cases. Histologically, the lesions were characterized by focal necrosis demarcated from surrounding normal tissue containing a number of clumps of bacteria and cellular debris. Bacteriological examination revealed that provocation of Coryne-bacterium kutscheri by ACTH-treatment resulted in appearance of the lesions. By means of intravenous or intraperitoneal inoculation with the strain isolated from lesion similar lesions were produced in the cecum of inoculated rats under the ACTH-treatment.     

In vivo toxicity and pulmonary effects of promazine and chlorpromazine in rats

Cationic amphiphilic drugs induce a phospholipid storage disorder known as phospholipidosis. Halogenated analogs of the drugs are more potent inducers of phospholipidosis when compared to nonhalogenated analogs. Two such antipsychotic drugs, promazine and chlorpromazine, are effectively taken up by the lungs and induce lamellar inclusions in vitro. We compared the in vivo toxicity and efficacy of promazine and chlorpromazine to induce phospholipidosis in the lung and in pulmonary alveolar macrophages. Male Sprague-Dawley rats were given promazine or chlorpromazine (25 mg/kg/day, P.O., in water) for 5 weeks. Food intake was decreased in promazine- and chlorpromazine-treated rats, chlorpromazine rats being affected more than promazine rats. To minimize experimental error due to starvation, control rats were pair-fed. The body weight gain was decreased in chlorpromazine rats in comparison to pair-fed controls. Chlorpromazine-treated rats, but not promazine-treated rats, showed increased mortality over the 5-week treatment period. Histopathologic examination of lung revealed loss of alveolar macrophages with no other gross abnormalities in chlorpromazine-treated rats. Quantitative analysis of lung lavage also showed significant reduction in the number of macrophages. This finding is in contrast to other cationic amphiphilic drugs, which induce phospholipidosis as well as accumulation of alveolar macrophages. Phospholipid level increased in alveolar macrophages but not in lavaged lung following chlorpromazine treatment. Acid phosphatase activity in lavaged lung homogenate and macrophages of promazine- and chlorpromazine-treated rats, taken as an index of toxicity to cells, did not differ significantly from control rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human galectin-3 is a novel chemoattractant for monocytes and macrophages

Galectin-3 is a beta-galactoside-binding protein implicated in diverse biological processes. We found that galectin-3 induced human monocyte migration in vitro in a dose-dependent manner, and it was chemotactic at high concentrations (1.0 microM) but chemokinetic at low concentrations (10-100 nM). Galectin-3-induced monocyte migration was inhibited by its specific mAb and was blocked by lactose and a C-terminal domain fragment of the protein, indicating that both the N-terminal and C-terminal domains of galectin-3 are involved in this activity. Pertussis toxin (PTX) almost completely blocked monocyte migration induced by high concentrations of galectin-3. Galectin-3 caused a Ca2+ influx in monocytes at high, but not low, concentrations, and both lactose and PTX inhibited this response. There was no cross-desensitization between galectin-3 and any of the monocyte-reactive chemokines examined, including monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha, and stromal cell-derived factor-1alpha. Cultured human macrophages and alveolar macrophages also migrated toward galectin-3, but not monocyte chemotactic protein-1. Finally, galectin-3 was found to cause monocyte accumulation in vivo in mouse air pouches. These results indicate that galectin-3 is a novel chemoattractant for monocytes and macrophages and suggest that the effect is mediated at least in part through a PTX-sensitive (G protein-coupled) pathway.     

鼠棒状杆菌PCR检测方法的建立及其应用

目的建立鼠棒状杆菌PCR检测方法并应用于临床样本检测。方法用脑心浸出液培养基复苏、培养鼠棒状杆菌（corynebacteriumkutscheri,C．kutscheri）并提取基因组DNA作模板;根据GenBank中C．kutsche6的16S基因序列设计合成引物,建立鼠棒状杆菌PCR检测方法并进行敏感性和特异性的评价;人工感染昆明鼠,建立小鼠棒状杆菌感染模型,采集肝脏和肾脏,提取DNA进行检测。结果成功建立了鼠棒状杆菌PCR检测方法,该方法可检测到100个阳性质粒;对小鼠沙门氏菌、肺炎链球菌和巴氏杆菌无交叉反应;全部8个人工感染样本全部检测为阳性。结论建立的鼠棒状杆菌PCR检测方法灵敏度高、特异性好,可作为鼠棒状杆菌感染的快速检测方法。