Germ-free Environment Research Articles

Spontaneous autoimmune diabetes mellitus in the BB rat.

The BioBreeding Rat is a recently discovered model of spontaneous diabetes mellitus. Studies to date have revealed the following characteristics of the syndrome: genetic predisposition, equal frequency and severity among males and females, absence of obesity, life sustaining requirement for insulin therapy, lymphocytic insulitis with destruction of pancreatic beta-cells, lymphocytic thyroiditis and the presence of autoantibodies to smooth muscle, thyroid colloid and other cellular antigens. Animals raised in a germ-free environment evidence diabetes with equal frequency and severity. Support for a cell-mediated autoimmune pathogenesis of the diabetic syndrome is derived from the following experiments: administration of antiserum to rat lymphocytes prevents diabetes in susceptible animals and normalizes plasma glucose levels in 36% of diabetic rats; neonatal thymectomy almost completely prevents the occurrence of diabetes. Although the BB rat may not be an appropriate model for studying the vascular complications of diabetes, peripheral nerve functional and ultrastructural defects have been reported and renal glomerular immuneglobulin deposits have been observed in long-term diabetic animals.

Early corneal changes in vitamin A deficient rats

Corneas of retinol deficient rats maintained on low levels of retinoic acid in a conventional laboratory environment and corneas from retinol deficient rats receiving no retinoic acid or retinoic acid for 5 weeks and kept in a germ free environment have been examined for structural abnormalities before the onset of apparent xerophthalmia. All groups of deficient rats showed abnormally large numbers of exfoliating cells, increased density of keratofibrils throughout the epithelial layer, decreased glycogen content, deposits of electron dense particles in the basal lamina region and accumulation of electron dense bodies in the keratocytes. These changes probably occurred as a primary effect of vitamin A deficiency. However, the absence of neovascularization and inflammation of the corneal stroma in the germ free rats, suggests that these changes may be secondary, perhaps due to infection.

Spontaneous diabetes in the gnotobiotic BB/W rat.

To determine the influence of infectious agents on the initiation of diabetes in the spontaneously diabetic Bio-Breeding/Worcester (BB/W) rat, susceptible rats were raised in a germ-free environment. Between 2 and 3 mo of age, 3 of 12 pups became diabetic. Histologic examination of the pancreas revealed insulitis or end-stage islets. Culture and smears from various tissues were negative for bacteria or parasites. Serum vital antibody titers for all known rat viruses were undetectable. These data suggest that the diabetic syndrome of the BB/W rat is not dependent on recognized infectious agents.

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.

Progressive depigmentation of the coat of C3H mice after neonatal thymectomy

Progressive depigmentation of the coat has been observed in C3H mice thymectomized at birth and reared in a germ-free environment. The origin of the pigmentary anomalies is discussed. It is probable that a causative relationship exists between thymectomy and the acquired depigmentation observed.

Immune responses of BALB/c mice to the idiotype of T15 and of other myeloma proteins of BALB/c origin: implications for an immune network and antibody multispecificity.

Normal adult BALB/c mice are virtually unresponsive to the idiotype of myeloma protein T15 when immunized with purified T15. However, antibodies to the T15 idiotype can be elicited by T15 in BALB/c mice that are reared in a germ-free environment or injected as newborns with an alloantiserum to the idiotype. Some germ-free and neonatally suppressed mice also produce helper cells (presumably T helpers) that enhance B-cell production of anti-DNP antibodies in response to DNP-T15. Taken together with previous studies, the present results mean that so far there are no exceptions to the rule that BALB/c mice have B and T cells that can respond to idiotypes of myeloma proteins of BALB/c origin. There appears to be a reciprocal relation between the natural prevalence of an immunoglobulin's idiotype and its immunogenicity in isologous individuals. The findings support proposals for an immune network of idiotypes and anti-idiotypes. Besides binding one or more extrinsic antigens, it is likely that each immunoglobulin also binds the idiotype of some other immunoglobulin. An immune network therefore implies multispecificity of antibodies.

Further Observations on the Effect of Reverse Isolation from Birth on Cognitive and Affective Development

Because he was considered at risk for combined immune deficiency disease an otherwise healthy boy was delivered by Caesarean section and placed at once in a germ-free environment. He has been maintained in reverse isolation since. The present report reviews aspects of his developmental progress during his first 52 months. Despite the highly atypical character of his living situation, he shows, at this time, no evidence of deviant emotional or intellectual development.

Motheaten: An Immunodeficient Mouse with Markedly Less Ability to Survive Than the Nude Mouse in a Germfree Environment

Summary The motheaten mouse is an immunodeficient mutant which has been found to have elevated immunoglobulin levels, to exhibit autoimmunity, and early death. Since survival is not improved by a germfree environment, it appears that some mechanism other than infection results in its early demise.

Allogeneic bone marrow chimerism in germ-free mice. IV. Therapy of "Hodgkin's-like" reticulum cell sarcoma in SJL mice.

"Hodgkin's-like reticulum cell sarcoma develops spontaneously in most SJL mice. Germ-free and conventional SJL mice bearing advanced reticulum cell sarcoma were treated with X-irradiation and transplantation of bone marrow from SJL or C3H/He donors. The 120-day survival rate of germ-free mice receiving allogeneic bone marrow (70%) exceeded that of all control groups; more importantly, germ-free mice that survived more than 4 months after treatment with allogeneic cells had no evidence of neoplastic lesions when killed. The germ-free environment effectively prevented graft-versus-host disease that was lethal to conventional mice. The results of these experiments offer additional evidence that bone marrow transplantation can be used as a therapeutic tool for spontaneous murine neoplasms.

Growth of human normal and neoplastic mammary tissues in the cleared mammary fat pad of the nude mouse.

Dysplastic and malignant human breast tissues were grown successfully in the cleared mammary fat pads (CFP) of nude mice. The mammary fat pads were cleared while the mice were in a germfree isolator. Prepared mice were removed fron the germfree enviornment to facilitate transplantation of the human mammary tissue into their CFP and subsequently were maintained in sterile laminar flow racks.

The germfree culture of three species of Triatominae: Triatoma protracta (Uhler), Triatoma rubida (Uhler) and Rhodnius prolixus Stål.

Triatoma protracta protracta, Triatoma rubida rubida, and 2 strains of Rhodnius prolixus were successfully reared germfree into the 4th generation. Triatomine eggs were disinfected externally with iodine and the nymphs reared in vinyl plastic gernifree modules. Gernifrce mice were used as hosts. This is the first report of Triatominae having been reared in a germfree environment. These bugs were never exposed to bacteria internally or externally to the 4th generation. Comparisons of conventional control bugs with replicate germfree groups in the first 2 generations revealed but slight differences in developmental periods. The mean adult longevity of all germfree groups was as long as, or longer than, that of the conventional adults. Fecundity and fertility of mated pairs were quite similar between germfree and control groups within a species. Evaluation of all differences in these various categories resulted in the conclusion that germfree Triatominae of these species develop in a normal manner in the absence of bacteria within or upon them when nourished by a suitable host (mouse). I found, as did Wigglesworth and others, that germfree Rhodnius prolixus did not develop to the adult stage when fed on a rabbit. The critical factor in the normal development of germfree R. prolixus evidently was the nutrition received from the mouse host. Nocardia rhodnii, Escherichia coli, and Streptococcus faecalis fed to germfree T. protracta resulted in the establishment of N. rhodnii and S. faecalis within the alimentary tracts of these bugs and the complete elimination of E. coli from the tracts of those bugs infected with this bacterium. When placed in a germfree module, bugs carrying S. faecalis and N. rhodnii readily transmitted these bacteria to previously germfree bugs maintained in the module. Bacillus thuringiensis var. thuringiensis fed to T. protracta and R. prolixus resulted in the death of all specimens ingesting this bacterial species.

Ascaridia galli (Nematoda): Development and survival in gnotobiotic chickens

Infection and growth rates of the fowl nematode Ascaridia galli were compared in germ-free, conventional, and other gnotobiotic chickens. Uniform experimental infections induced in chickens at 7–12 days of age showed that significantly fewer ( P < 0.01) worms established themselves in germ-free chickens than in chickens with conventional flora. Numbers of worms established in monoassociated hosts ( Bacillus subtilis, B. cereus, and Penicillium sp.) were intermediate between those in grossly contaminated and germ-free hosts. Mean worm lengths were shorter from germ-free than conventional hosts but differences were not statistically significant. These experiments demonstrate that some A. galli larvae may establish themselves and grow for 14 or 21 days postinoculation in a germ-free environment.

The role of maternal effects in animal breeding. I. Passive immunity in newborn animals.

The Why of Passive Immunity WHY is it that newborn animals require a supply of gamma globulin passively acquired from their maternal parent? Certainly it is not that they cannot make their own antibodies; indeed, many can. Studies in recent years have shown that developing fetuses can make antibodies to numerous antigens but, in the isolation of their essentially germ-free environment, they rarely come in contact with foreign antigens. The fetal lamb when exposed to various antigens first makes antibodies to bacteriophage φ174, later to ferritin and hemocyanin, and still later to ovalbumin but not at all to diptheria toxoid (Sterzl and Silverstein, 1967). Those studies and comparable studies on embryonating chicks (Brambell, 1970) demonstrate that immunological competence does not arise simultaneously for all antigens. Moreover, competence continues to mature post-term, and it becomes greatly enhanced by multiple contacts with literally hundreds of different antigens. Thus, the logical inference is that passive immunity is required because the

The influence of the microflora of the alimentary tract on protein digestion in the chick.

1. The course of digestion of freeze-dried and heat-damaged egg albumen labelled with14C in all its constituent amino acids was compared in germ-free and conventional chicks. The albumen was given at a level of 10% in a test meal containing 28% protein. Samples of digesta were taken 5 h later and analysed for14C and nitrogenous constituents.2. After administration of unheated albumen, 11% of the14C remained in the germ-free gut, compared with 23% in the conventional gut. The poorer digestibility of the heat-damaged albumen was shown by recovery of 42 and 47% of the14C from the gut of germ-free and conventional birds respectively.3. With both diets there was a higher14C: nitrogen ratio in the contents of the lower gut of conventional birds. Most of the N remaining in the gut was composed of protein or the hydrolysis products of protein, the amounts of which tended to be higher in the germ-free environment; urea and uric acid formed only a small proportion of the total N. There was less urea and more uric acid in the lower gut and excreta of conventional chicks.4. It was concluded that microbial activity did little to increase the availability of protein to the chick. However, its effect on the nitrogenous material in the lower part of the alimentary tract might result in low recovery of N in the faeces and thus lead to erroneously high estimates of protein quality in tests with conventional chicks.

A comparison of the effects of raw and heated soya-bean meal in diets for germ-free and convetional chicks.

1. Chicks were reared to 3 weeks in a germ-free or conventional environment on diets containing 50% raw or heated soya-bean meal. Body-weights and pancreas weights were determined and the proteolytic and amylolytic enzymes in the pancreas were measured. In one experiment the contents of the intestinal tract were analysed for their content of nitrogenous compounds.2. The growth of chicks given raw soya was depressed below that of controls given heated soya, and the growth depression was significantly greater in the conventional compared with the germ-free environment.3. The pancreases of chicks given raw soya were significantly larger than those given heated soya. The degree of enlargement was similar in both environments. The concentration of α-amylase was lower in the enlarged pancreases, but the total amount per pancreas was similar to that in the normal pancreases. Conversely, the concentration of proteolytic enzymes was the same or greater in the enlarged pancreases, which had significantly greater total content of proteases than did the normal pancreases.4. There was evidence of impaired protein digestion in the birds given the raw soya diets. Their gut contents contained larger amounts of insoluble matter, with higher nitrogen content, and the soluble fraction contained more nitrogen present as ‘protein’, with correspondingly less ‘peptide’ and ‘amino acid’, than did similar samples from birds given heated soya. No differences were observed in this respect between germ-free and conventional birds.5. It was concluded that the gut microflora exacerbated the growth-depressing effect on chicks of raw soya meal. Possible explanations are advanced.

Experimentally induced changes in nasal mucous secretory systems and their effect on virus infection in chickens. I. Effect on mucosal mrphology and function.

The domestic chicken was used as an experimental model in which to demonstrate morphological and functional relationships of nasal organ systems, principally of mucous systems. Mucous secretions of olfactory, respiratory, lacrimal, and accessory areas were found to have clear histochemical differences, yet were sufficiently miscible in normal circumstances to form an unbroken, synchronously moving sheet. Changes induced experimentally in host physiology did not all affect the mucous components of given areas in the same way or to the same degree. Mucosal changes were produced by the following methods: Topically administered cocaine 20%, in a single application, temporarily paralyzed the cilia, and the consequently reduced traction apparently held mucus in the acini and effected a temporary lag in mucus excretion. Three successive applications caused acute acinar depletion and ciliary paralysis. Hexylcaine chloride 5% immediately desquamated all intranasal epithelia, damaged the proximal portion of the acini, and induced acinar exhaustion and mucosal inflammation-effects not overcome within 5(1/2) days. Internal dehydration produced progressively viscous mucus, severe acinar gaping with mucus anchored in the acini, a heavy surface sheet, and deceleration or arrest of mucociliary flow. Avitaminosis A induced reduction in the height (about 50%) of all mucosae and acini, especially the inner lining of the maxillary concha, caused an actual 50% reduction in the number of cells per acinus, and retarded the mucociliary flow rate about 50%. Pilocarpine induced initial hypersecretion, later exhaustion, and, still later, slow production of densely staining mucus in the acinar cells; also acinar gaping. Breeding in a germfree environment produced a greatly reduced mucosal depth throughout the nasal fossa, an extraordinary reduction in the number of cells per acinus, relative reduction in the number of acinar neck cells, and concomitant increase in ciliated cells in that region. Exposure to a temperature of -20 degrees C for 1 hr caused blanching of all secretory cells, acinar gaping, and temporary reduction of mucosal depth.

The use of an isolator system in cancer chemotherapy

A patient isolator system was employed to evaluate the effect of reduction of normal flora and of a relatively germ-free environment upon patients receiving myelosuppressive therapy. Twelve patients with a variety of malignancies underwent intensive chemotherapy within the isolator for a total of 537 days. The average period of occupancy was forty-five days. Almost all patients were considered treatment failures prior to therapy within the isolator. There were five patients with choriocarcinoma, four with acute leukemia and three with chronic myelogenous leukemia in blastic crisis. The antimicrobial regimen directed at reducing the patients' bacterial flora was reasonably effective and resulted in 60 per cent sterile stool cultures. The intensive chemotherapy administered resulted in granulocyte counts less than 500 per cu. mm. on 53 per cent of patient days. The incidence of infection was about 30 per cent of that which might have been expected outside the isolator. Observed infections included two gram-negative septicemias, one thrombophlebitis, one cellulitis and one case of multiple pulmonary abscesses. Of the five patients with choriocarcinoma, chorionic gonadotrophic hormone titer was reduced in three, unchanged in one and increased in one. All the patients with acute leukemia had remissions ranging from two to seven months with an average of five months. The three patients with chronic myelocytic leukemia showed no improvement.

Prevention of Infectious Diseases in Experimental Mice.

SummaryBy employing conventional husbandry techniques and filter-capped cages, a breeding colony of mice from Caesarian-derived stock was maintained free from many infectious agents common to laboratory mice through seven generations. The system described prevented infections with pathogenic bacteria, viruses, parasites, and PPLO. Mice housed under these conditions remained healthy and fecund while being free of the morphological and physiological consequences of germfree environment. In addition, the expense and restrictions of conventional barrier-type operations were minimized.

Granulocytopoiesis in germfree mice.

SummaryA comparison of the total number of neutrophils and neutrophil precursors within the humerus of germfree and conventional mice revealed no statistically significant difference between the two groups. The distribution of marrow neutrophils with respect to morphologic type and the number of neutrophils in mitosis were similar in the two groups. There was, however, a slight, but significantly, lower concentration of blood neutrophils in germfree mice as compared to controls. Removal of mice from the germfree environment was associated with a rapid decrease in the number of mature and relatively mature neutrophils in the marrow and with a subsequent increase in potentially mitotic, immature neutrophils, following which the marrow returned to normal. Germfree mice responded to endotoxm administration with an increase in rate of release of neutrophils from the marrow, as do normal mice. It was concluded that in this model system the presence or absence of microorganisms in the environment could not be as...

AUTOIMMUNE REACTIONS AND VIRUS-LIKE PARTICLES IN GERM-FREE NZB MICE

Autoimmune reactions, as shown by positive antiglobulin (Coombs) tests and the presence of antinuclear factors in the serum, occur in NZB mice bred and maintained in a germ-free environment. Serum-immunoglobulin levels are unaltered despite this isolation from exogenous antigenic stimuli. Particles resembling murine-leukæmia virus are found in germ-free NZB mice as in their conventional counterparts indicating transmission via the germ cells or placenta. The presence of virus-like particles might be associated with maintenance of serum-immunoglobulin levels and the development of autoimmune reactions; but more positive evidence is needed before this association can be regarded as more than fortuitous.