Cytokine responsiveness in germfree and conventional NMRI mice

We have investigated the proliferative response of thymocytes from different mouse strains to cytokines in vitro. Interleukin 2 (IL-2), IL-4 and IL-7 induced proliferation of thymocytes from NMRI/KI (a locally bred NMRI mouse strain), NMRI/H ('traditional' NMRI mice), C3H/HeJ and C3H/HeN mice. NMRI/KI thymocytes showed the most prominent proliferation in response to IL-1 alpha and IL-1 beta. IL-3, IL-5, IL-6, IL-8, IL-10, tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), inhibin and lipopolysaccharide (LPS) induced no thymocyte proliferation. Germfree NMRI/KI mouse thymocytes showed a significantly lower proliferation in response to IL-1 alpha and IL-1 beta than conventional mice. Rat tissues, previously shown to contain lymphocyte activating factors (LAFs), were also tested. Skin, tongue, esophagus, proventricular stomach, testis and placenta were all positive in the LAF assay utilizing NMRI/KI thymocytes, whereas none of the tissue extracts could induce proliferation in NMRI/H thymocytes. The higher cytokine responsiveness in conventional mice compared with germfree might suggest that exposure to microflora induces a higher state of activation of the immune system. The LAF assay, utilizing NMRI/KI thymocytes, is a highly sensitive IL-1 bioassay with a detection level of 1 pg/ml for IL-1 beta and 2 pg/ml for IL-1 alpha. The specificity of the assay is increased by utilizing NMRI/H mice to exclude the presence of IL-2, IL-4 and IL-7.     

Differences in survival among germfree mice following transfer to a conventional colony.

Sex and strain differences in survival were studied in 7-9 month old germfree mice following transfer to a conventional colony. One outbred and three inbred strains were observed. All outbred CD-1 mice survived transfer and in 4 months increased their weight by 50%. The majority of inbred mice survived 7 months after transfer. Sex differences in survival were evident throughout the experimental period and were most marked 7 months after transfer. An unexpected new finding was the viability of the male sex in germfree mice after transfer. Possible explanations are considered.     

Mycoplasmosis in specific-pathogen-free and conventional guinea pigs.

Normal female guinea pigs were examined for organisms of the order Mycoplasmatales. Thirty-nine isolations were made from nose or vagina of 108 guinea pigs. Seven isolates were identified as Mycoplasma caviae, 9 were unidentifiable mycoplasmas, and 23 were identified as nonsterol-requiring acholeplasmas.     

Buoyant density analysis of myeloid colony-forming cells in germfree and conventional mice.

Granulocyte-macrophage colony-forming cells (CFUc), in the bone marrow of germfree and conventioal CBA mice, were compared quantitatively and qualitatively. Cells were separated on the basis of their buoyant density by equilibrium centrifugation in continuous albumin density gradients. CFUc in the density subpopulations were detected by culture in agar containing three different types of colony stimulating factor (CSF). The sources of the CSF were post-endotoxin mouse serum (CSFES), mouse lung conditioned medium (CSFMLCM) and human urine (CSFHU). Mice were removed from the germfree environment and the buoyant density status of their CFUc was examined 1, 4 and 8 weeks later. No difference was found between germfree and conventional mice in the number of nucleated cells per femur or in their modal density. Neither was the number of CFUc per femur different. The cell cycle status of CFUc, as determined by the thymidine suicide technique was not significantly different. Functional heterogeneity was found among the density subpopulations for both groups of mice. This depended on the type of CSF. The density distribution of CFUc was significantly different in germfree mice. There were proportionately more low density CFUc. The mean modal density of CFUc under CSFES stimulation was less by 0.0045 g/cm3 in germfree mice. The removal of mice from the germfree environment resulted in a shift of the distribution to higher densities. The trend was towards the conventional situation. The significance of the buoyant density status of CFUc is discussed.     

Serum amyloid A, cytokines, and corticosterone responses in germfree and conventional mice after lipopolysaccharide injection.

To determine why germfree mice are less susceptible to lipopolysaccharide (LPS) than conventional mice, we studied serum levels of serum amyloid A (SAA), tumor necrosis factor (TNF), interleukin 1 (IL-1), IL-6, and corticosterone in mice after treatment with LPS. A single injection of LPS caused an elevation of SAA, an acute-phase protein in the mouse, in both conventional and germfree IQI mice, and the response was significantly less in germfree mice. LPS-induced elevations of serum TNF, IL-1, and IL-6 levels were also significantly less in germfree mice, while serum corticosterone levels were greater in germfree mice than in conventional mice. These results suggest that the lower susceptibility to LPS and a smaller response of SAA elevation by LPS in germfree mice may result from less elevation in serum of these cytokines in these mice, which are known to mediate the acute phase response of SAA. High levels of serum corticosterone in germfree mice may be partly responsible for the lower responsiveness of these inflammatory cytokines to LPS in these mice.     

Comparative studies of some functional and morphological parameters in the livers of germfree, conventional and ex-germfree mice.

Some parameters of hepatic function and morphology were studied to compare germfree (GF) and conventional (CV) BALB/c mice. The levels of lipid peroxide (LPO) and aniline-hydroxylase (AH) activity in the livers and the serum total cholesterol (TC), triglyceride (TG) and phospholipid (PL) were significantly lower in GF than in CV 8-week-old mice. There were no significant differences in the histology and lectin-histochemistry of the livers in the GF and CV mice. On the other hand, in ex-GF mice which were induced by housing 4-week-old GF mice together with age-matched CV mice, the levels of LPO and AH activity in the liver and the serum TC, TG and PL contents increased rapidly within the first week and then approached values almost identical to those in CV mice 4 weeks later (i.e. at 8 weeks of age). The histologic picture of the liver was similar among the GF, CV and ex-GF mice.     

Retention of ingested latex particles in Peyer's patches of germfree and conventional mice

Conventional and germfree mice ingested a suspension of 2-micron latex particles in drinking water for a 15-day period. Number and distribution of intestinal Peyer's patches did not differ significantly in the two types of mice. Cleared Peyer's patches were compared with regard to size and particle content. The location of particles within Peyer's patch follicles of germfree mice was similar to that of conventional mice, but the latter had significantly larger follicles and greater accumulations of latex particles. Latex concentration varied with patch location. Proximal patches contained the majority of particles in germfree mice, whereas particles were most abundant in distal patches of conventional mice. The results show that particle uptake into Peyer's patches takes place even in the complete absence of bacteria in the gut.     

Analysis of bile acids in conventional and germfree rats.

The well-known bile acid analysis technique used by us and others (Grundy, Ahrens, and Miettinen. 1965. J. Lipid Res. 6:397-410) does not allow for the detection of hyodeoxycholic acid, a product of quantitative importance in rodent feces. Using updated methodology, it was established that hyodeoxycholic acid and omega-muricholic acid, both apparent conversion products of beta-muricholic acid, occur in apppreciable amounts in intestinal contents and feces of conventional Wistar type Lobund rats. In conventional rats, these bile acids comprise about 50% of fecal bile acids; they are not found in intestinal contents or feces of germfree rats. Others have demonstrated that hyodeoxycholic acid if formed by combined action of gut flora and liver. A new method for the separation of conjugated and free bile acids in biological samples was developed. Results with this method confirmed the total conjugation of bile acids in the germfree rat, and the almost total deconjugation that takes place in the cecum of the conventional rat.