C. parvum in germ-free mice

Summary The effect of previous sensitization to C. parvum, by cross-reacting antigens from other bacteria, on the immunostimulatory effects of C. parvum treatment were studied in germ-free and conventional mice. It was found that the development of splenomegaly and specific delayed hypersensitivity following C. parvum injection were similar in both germ-free mice and conventional mice.Supported by U.S. Public Health Service Grant No. 5S07 RR05705 and NIH Grant no. AM 18530Visiting Investigator. Present address: Department of Experimental Immunobiology, The wellcome Research Laboratories Beckenham, Kent, England.Recipient of a post-doctoral fellowship from the National Foundation for Ileitis and Colitis Inc.Recipient of Research Career Development Award No. AM 0073 from the National Institute of Arthritis, Metabolism and Digestive Diseases     

Use of germ-free mice in experimental transplantation

A comparative study of skin allografts was performed in germ-free A/Ola mice kept in boxes and in A/Ola and BALB mice raised in ordinary conditions. Skin graft (of C57B1/6 mice) in A/Ola and BALB mice raised in ordinary conditions was shown to reject 16-21 and 12-18 days after the transplantation, respectively without cyclophosphamide (CP) use. CP application in BALB mice, grown in ordinary conditions, prolonged the lifespan of grafts to 12-29 days. The use of CP in germ-free A/Ola mice prolonged the lifespan of grafts to 19-39 days. In germ-free mice kept in boxes the use of an immunodepressant was not accompanied by infectious complications, while the animals kept in the vivarium often died of infectious diseases.     

Detection of viruslike particles in germ-free mice

Kajima, Masahiro (University of Notre Dame, Notre Dame, Ind.), and Morris Pollard. Detection of viruslike particles in germ-free mice. J. Bacteriol. 90:1448-1454. 1965.-Viruslike particles similar in structure to the mouse leukemia agent were detected by electron microscopy in thymus tissues of germ-free AK mice which had developed spontaneous leukemia. In addition, unique viruslike particles (type B) were detected in tissues from spontaneous mammary adenocarcinoma of germ-free C3H mice. Leukemia virus-like particles were also observed in the thymuses of the control AK mice and of the C3H control mice as well as of those with mammary tumors. Germ-free mice are not virus-free. The routes of transmission of leukemia as well as of mammary tumor viruses may be "vertical," through the embryo or placenta.     

Alterations in brain lipid composition of mice made physically dependent to ethanol

The ability of chronic ethanol treatment to alter CNS membrane lipids was tested. Adult male C57/BL6 mice were given a liquid diet containing ethanol for eight days. This regimen produced strong physical dependence as judged by withdrawal seizures, tremors and concomitant hypothermia. Analyses were performed on cholesterol, total phospholipid content and total phospholipid acyl composition of myelin, crude (P2), light and heavy synaptosomes as well as synaptosomal plasma membranes. Chronic ethanol treatment had no effect on total phospholipid levels nor phospholipid acyl composition in any of the above subcellular fractions. In ethanol dependent mice, significant increases in cholesterol content and cholesterol/ phospholipid ratios were observed only in synaptosomal plasma membranes.     

Decreased cellular immune response of germ-free mice.

Cellular immune response to intracerebral lymphocytic choriomeningitis infection was studied in mice belonging to an identical strain but different in breeding conditions. In consequence of the cellular immune reaction on the leptomeninx, lymphocytic choriomeningitis developed and caused death in 100% of conventionally bred mice, whereas 80% of germ-free and 15% of mouse-pathogen-free mice failed to display lymphocytic infiltration of the leptomeninx and survived the infection as chronic virus carriers. This finding pointed to a deficient cellular immune response of germ-free and mouse-pathogen-free mice. The under-development of the lymphoid system due to the antigen-poor breeding conditions might be responsible for the deficiency.     

Alterations of beta-adrenergic signaling and cardiac hypertrophy in transgenic mice overexpressing TGF-beta(1)

Transforming growth factor-beta(1) (TGF-beta(1)) promotes or inhibits cell proliferation and induces fibrotic processes and extracellular matrix production in numerous cell types. Several cardiac diseases are associated with an increased expression of TGF-beta(1) mRNA, particularly during the transition from stable cardiac hypertrophy to heart failure. In vitro studies suggest a link between TGF-beta(1) signaling and the beta-adrenergic system. However, the in vivo effects of this growth factor on myocardial tissue have been poorly identified. In transgenic mice overexpressing TGF-beta(1) (TGF-beta), we investigated the in vivo effects on cardiac morphology, beta-adrenergic signaling, and contractile function. When compared with nontransgenic controls (NTG), TGF-beta mice revealed significant cardiac hypertrophy (heart weight, 164 +/- 7 vs. 130 +/- 3 mg, P < 0.01; heart weight-to-body weight ratio, 6.8 +/- 0.3 vs. 5.1 +/- 0.1 mg/g, P < 0.01), accompanied by interstitial fibrosis. These morphological changes correlated with an increased expression of hypertrophy-associated proteins such as atrial natriuretic factor (ANF). Furthermore, overexpression of TGF-beta(1) led to alterations of beta-adrenergic signaling as myocardial beta-adrenoceptor density increased from 7.3 +/- 0.3 to 11.2 +/- 1.1 fmol/mg protein (P < 0.05), whereas the expression of beta-adrenoceptor kinase-1 and inhibitory G proteins decreased by 56 +/- 9.7% and 58 +/- 7.6%, respectively (P < 0.05). As a consequence of altered beta-adrenergic signaling, hearts from TGF-beta showed enhanced contractile responsiveness to isoproterenol stimulation. In conclusion, we conclude that TGF-beta(1) induces cardiac hypertrophy and enhanced beta-adrenergic signaling in vivo. The morphological alterations are either induced by direct effects of TGF-beta(1) or may at least in part result from increased beta-adrenergic signaling, which may contribute to excessive catecholamine stimulation during the transition from compensated hypertrophy to heart failure.     

Determining the viability of faecal bacteria present in germ-free mice

Gram-positive bacilli, originating from the diet, are present in the faeces of germ-free mice in this Unit. Although these organisms have never grown on culture and are assumed to be dead it was considered desirable to test this by non-cultural methods because some gut bacteria are difficult or impossible to grow in vitro by present techniques. Germ-free mice were fed a synthetic diet free from live or dead organisms for 10 days during which time the bacilli disappeared from the faeces, re-appearing when the usual diet was re-introduced. This was regarded as confirmation of the non-viability of the bacilli. The use of Wayson's stain for confirmation of non-viability was found to be inappropriate since it produced false-positive reactions with irradiation-killed bacteria.     

Variations of brain histamine levels in germ-free and nephrectomized rats

The influence of nephrectomy on brain and peripheral tissue histamine and on brain norepinephrine, dopamine, serotonin, and 5-hydroxyindoleacetic acid was studied in germ-free and conventionally housed rats. The conventional controls had higher levels of histamine in the hypothalamus than the germ-free control animals, but no differences existed for histamine in whole brain minus the hypothalamus or in peripheral tissues. Nephrectomy increased brain histamine and 5-hydroxyindoleacetic acid levels in both germ-free and conventional rats, but had no effect on norepinephrine, dopamine or serotonin. In contrast, the histamine level in the heart of the nephrectomized germ-free animals was lower than that for germ-free controls. There were no changes in the heart or liver histamine levels of the conventional nephrectomized rats.     

An improved procedure for obtaining germ-free laboratory mice

The economy of time and effort accruing from the use of laminar air-flow cabinets to obtain germ-free mice has already been established. However, a further modification of the technique, utilizing small intermediate isolators, has made it possible to transfer to the large, recipient isolators only those litters which are viable and demonstrably free from contamination.     

Alterations of microRNAs expression profiles in small extracellular vesicle after traumatic brain injury in mice

Traumatic brain injury (TBI) is one of the leading causes of mortality and morbidity worldwide. Tools available for diagnosis and therapy are limited. Small extracellular vesicle (sEV) microRNAs (miRNAs) play an important role in TBI disease progression. This study aimed to investigate the alterations in sEV miRNAs expression in the mouse brain extracellular space after TBI. Twenty-four C57BL/6J mice were randomly divided into two groups (12/group). The TBI group was subjected to all surgical procedures and fluid percussion injury (FPI). The sham group only underwent surgery. Brain specimens were collected 3 h after TBI/sham. The brain sEV were isolated. Differentially expressed miRNAs were identified. A total of 50 miRNAs were observed to be differentially expressed (fold change ≥1.5 and P<0.05) after TBI, including 5 upregulated and 45 downregulated. The major enriched Gene Ontology terms were metabolic processes, cell, intracellular, organelle, cytoplasm, axon, binding, protein kinase activity, protein binding, and protein dimerization activity. The KEGG pathway analysis predicted that the pathways affected by the variation of miRNAs in sEVs after TBI included the Wnt signaling pathway and NF-κB signaling pathway. The changes in five miRNAs were confirmed by qRT-PCR. In conclusion, this study demonstrated the differential expression of a series of miRNAs in brain sEV after TBI, which might be correlated with post-TBI physiological and pathological processes. The findings might also provide novel targets for further investigating the molecular mechanisms underlying TBI and potential therapeutic interventions.     

Monocontamination of germ-free mice by a fastidious unidentified anaerobe

Over a period of several months, 5 germ-free mouse isolators became monocontaminated with seemingly the same fastidious, anaerobic bacilli which defied identification. The organism originated in rodent diet which had been exposed to gamma-irradiation. However, it was discovered retrospectively that the irradiation doses had not been monitored, and subsequent monitoring showed the desired dose of 5 Mrad was not achieved in some parts of the load. Although inadequate sterilization was the likely cause of contamination it was not possible to explain why only this fastidious anaerobe survived the treatment, and not other more common bacterial contaminants.