Gastrointestinal decontamination of dogs treated with total body irradiation and bone marrow transplantation.

Procedures for total and selective gastrointestinal decontamination of dogs are described. The selective procedure removed only Gram negative aerobic bacteria, yeast and fungi. Dogs receiving total decontamination were less susceptible to the GI syndrome following total body irradiation (TBI) than dogs receiving conventional care. After TBI and allogeneic bone marrow transplantation, serum albumin levels decreased in conventional animals, but remained normal in totally or selectively decontaminated animals. Exogenous infections occurred frequently in both irradiated, and totally decontaminated animals, but were absent in selectively decontaminated animals. Endogenous infections after total body irradiation were prevented only by total decontamination. Endogenous infections occurred in selectively decontaminated animals, but with milder clinical symptoms than in conventional animals. Appearance of donor type leukocytes and serum gamma globulin was slower in decontaminated animals than in conventionally treated controls. Acute graft versus host disease caused by a limited number of lymphocytes of a DLA identical littermate donor were prevented by selective gastrointestinal decontamination. Complications due to late immune reconstitution obscured the effect of decontamination on delayed graft versus host disease.

Quantification of antigen-specific immunoglobulin A after oral booster immunization with ovalbumin in mice mono-associated with segmented filamentous bacteria or Clostridium innocuum.

Segmented filamentous bacteria (SFB) are known to stimulate the mucosal immune system. Here, the effect of SFB on oral booster immunization with ovalbumin was investigated. Mice mono-associated with SFB or Clostridium innocuum were sensitized by intraperitoneal administration of 100 micrograms ovalbumin with Freunds complete adjuvant. After 4 weeks, mice received 80 mg ovalbumin orally. A maximum IgA response was found 5 days after this booster immunization. Comparison of mice with SFB and mice with C. innocuum revealed a much higher level of IgA in the gut lumen and more IgA secreting cells in the lamina propria of the SFB-associated mice. However, no differences between both groups of animals were found in specific levels of IgA secreting cells or luminal IgA against ovalbumin. It is concluded that there is no enhancing effect of SFB after booster immunization when mice are primed intraperitoneally with ovalbumin.

IFN-gamma-mediated prevention of graft-versus-host disease: pharmacodynamic studies and influence on proliferative capacity of chimeric spleen cells.

Recently we demonstrated that prolonged administration of IFN-gamma prevented the development of GVHD in a MHC-mismatched murine BMT model. Treatment with IFN-gamma allowed the development of mature donor-derived allo-tolerant immunocompetent cells in complete chimeric recipients. Here we present data on the pharmacodynamics of this cytokine-mediated protection against GVHD. Treatment with 50000 U IFN-gamma twice weekly for a period of 5 weeks, starting at the day of BMT, was shown to be the optimal treatment protocol, resulting in complete prevention of GVHD-related mortality. Treatment during 1 week with a three-fold higher weekly dose of IFN-gamma (50000 U six times) did not result in significantly improved survival. The start of IFN-gamma administration was a critical factor since a delay of 3 days from the time of BMT resulted in substantial GVHD-induced mortality. Furthermore, it was shown that IFN-gamma treatment inhibited the spontaneous and Con-A-induced proliferation of T cells at 7-14 days after BMT, which is the critical period for the initiation of acute GVHD. However, long-term survivors after IFN-gamma treatment showed a recovery of immunity in contrast to long-term survivors of saline-injected animals, as tested by Con-A responsiveness. It seems that injection of high dose IFN-gamma suppresses the response of potentially alloreactive donor T cells during what normally is the initiation phase of the GVH reaction (GVHR), resulting in the abrogation of GVHD.

Interferon-gamma-mediated prevention of graft-versus-host disease: development of immune competent and allo-tolerant T cells in chimeric mice.

Recently it was shown that delayed graft-versus-host disease (GVHD) in mice can be completely prevented by repeated injections of interferon-gamma (IFN-gamma). The characteristics of this sustained IFN-gamma-induced chimerism were studied in more detail. First, the potency of IFN-gamma as a modulator of GVHD was tested in a fully H-2 mismatched murine bone marrow transplantation (BMT) model. Donor bone marrow cells (BMC; C57BL/Rij; H-2b) were mixed with increasing numbers of donor spleen cells (SC) and transplanted into lethally irradiated recipients (C3H/Law; H-2k). Secondly, BMC and SC of the IFN-gamma-induced chimeras (C3H/Law; H-2b) were tested on their immunological competence and GVHD inducing capacity. Repeated injections of the host with IFN-gamma were able to prevent GVHD even when up to 10(5) SC were added to the graft; adding higher numbers of SC resulted in a rapid increase in the frequency of lethal GVHD. Donor-derived lymphocytes (H-2b) obtained from chimeric animals were immunocompetent as concluded from Con A stimulation in vitro. Chimeric-derived BMC (H-2b) were mixed with up to 10(7) chimeric SC (H-2b) and transplanted into a new group of lethally irradiated C3H/Law (H-2k) recipients. All transplanted animals survived the latter treatment without any macroscopic signs of histological lesions typical of GVHD. We conclude that IFN-gamma treatment allows the development of mature donor-derived immunocompetent T cells, which are allo-tolerant for the recipient.

Background (spontaneous) immunoglobulin production in the murine small intestine before and after weaning.

The ontogeny of the murine intestinal B-cell compartment before and after weaning was studied by quantitative analysis of immunoglobulin-secreting cells (Ig-SC) in the small intestine (SI). Before weaning, few Ig-SC were detected in the SI, whereas spleen and bone marrow already contained many Ig-SC. The number of Ig-SC in the SI started to increase immediately after weaning. Comparing early-weaned mice with non-weaned mice of the same age clearly demonstrated that weaning brought on the development of Ig-SC in the SI. The influence of a gut flora on the number of Ig-SC in the SI was examined by comparing the number of Ig-SC in the SI of conventionally housed, specific pathogen free (SPF) and germ-free mice. A bacterial flora was apparently needed for the normal development of Ig-SC in the SI. Comparing mice containing an aerobic Gram-negative bacterial flora with mice containing only an anaerobic Gram-positive bacterial flora demonstrated that the type of bacterial flora is relatively unimportant. No evidence was found that circulating maternal antibodies suppressed the development of the "spontaneous" intestinal and systemic B cell response. The results show that bacterial colonization of the intestine plays a pivotal role in the development of the Ig-SC compartment in the SI.

Detection of total- and partial-body irradiation in a monkey model: a comparative study of chromosomal aberration, micronucleus and premature chromosome condensation assays.

PURPOSE: To investigate the efficacy of three cytogenetic methods (dicentrics, micronuclei (MN) and premature chromosome condensation (PCC) analysis) for assessment of the unirradiated fraction and the persistence of damage after total-body (TB) and partial-body (PB) irradiation of rhesus monkeys (Macaca mulatta). MATERIALS AND METHODS: Animals were exposed to X-rays (5 Gy), either TB or PB, with about 6% of marrow cells shielded. Blood samples were collected at different times after exposure, i.e. 1, 3 and 7 days, and cultures were set up for the different cytogenetic endpoints. In addition, blood count analysis was performed before and after irradiation. RESULTS: Blood count analysis was not suitable for discriminating between TB and PB exposure. By using Poisson or overdispersion distribution as the basis, it was not possible to distinguish TB from PB irradiation when dicentric chromosomes and MN were analysed. PCC analysis, in contrast, showed a Poisson distribution after TB exposure and overdispersion after PB exposure. Using the PCC assay, reliable dose estimates could be obtained up to 7 days after irradiation. CONCLUSIONS: For dicentrics and MN, shielding of 6% of bone marrow cells was found to be too small to estimate the unirradiated fraction accurately. The PCC technique was useful for dose assessment and the inhomogeneous exposure of 6% was detected within a short period of time after exposure.

The use of a rat-derived microflora for providing colonization resistance in SPF rats.

To obtain a suitable species-specific microflora for a new rat SPF-unit, germ-free WAG/Rij rats were associated with a flora derived originally from selectively decontaminated Cpb: WU (Wistar) rats. Caecal and ileal contents of these rats had been cultured anaerobically (37 degrees C) for 7 days and harvested. This cultured flora was given to germ-free Cpb: SE (Swiss) mice, which were kept in an isolator system and acted as a source of the flora to associate germ-free Wag/Rij rats. In these associated rats, several parameters indicative of the 'quality' of the intestinal microflora were investigated and compared to those in rats with a mouse derived anaerobic microflora. Parameters included relative caecal weight, colonization resistance and the concentration of faecal bile acids. The cultured rat-derived microflora normalized the observed intestinal parameters better than the mouse derived microflora, and provided better colonization resistance. We conclude that culturing of intestinal contents of selectively decontaminated animals can be a useful way to obtain a species-specific donor-microflora which can be used to start new SPF units.

[Nalidixic acid intoxication in two pregnant bitches (author's transl)]. / Nalidixinezuur intoxicatie bij twee drachtige teven.

To ensure prolonged survival, dogs with cyclic neutropenia should be protected against bacterial infection of exogenous or endogenous origin, particularly during the neutropenic episodes. One of the methods available to minimize the risk of infection in these dogs, is selective decontamination of the gastrointestinal tract by using antibiotics and/or chemotherapeutic agents, in conjunction with housing in a laminar-flow cabinet. Two pregnant bitches, some of the offspring of which were expected to be homozygous for the cyclic neutropenia allele, were decontaminated with nalidixic acid. Fourteen days after initiation of the antibacterial treatment, the two dogs died. Jaundice and seizures had been apparent in both animals prior to death. Histopathological examination revealed changes primarily in the liver, which were consistent with toxic hepatic necrosis and were characterized by severe centrilobular haemorrhage and disappearance of hepatocytes. Multiple haemorrhages were observed in other organs. Further clinical investigation in two other dogs strongly suggested that nalidixic acid was the cause of death in the two pregnant bitches.

Management of bacterial and fungal infections in bone marrow transplant recipients and other granulocytopenic patients.

A review of studies on the effect of different types of gastrointestinal decontamination and protective environment on infectious complications in granulocytopenic patients is given, and the effect of these measures on graft-versus-host disease after allogeneic bone marrow transplantation is discussed. It is concluded that complete gastrointestinal decontamination of patients nursed under conditions of strict reverse isolation will maximally prevent infections, graft-versus-host disease, and lung complications and therefore is the treatment to be preferred for patients undergoing bone marrow transplantation. Since selective decontamination is as effective in preventing bacterial and fungal infections as is complete decontamination, this treatment is to be preferred for other patients with a greatly reduced resistance to these infections. The reason is that, for this type of patient, selective decontamination can be performed without the use of strict isolation facilities and in this way is less laborious and less of a burden for the patient. Besides this, the number of patients that can be treated will not be limited by the number of available facilities for strict reverse isolation, which can be reserved for bone marrow transplant patients.

Bacteriological testing of a modified laminar flow microbiological safety cabinet.

A modified microbiological safety cabinet which can be used as a class II and a class III safety cabinet has been bacteriologically tested. This cabinet makes use of a high-speed down-flow air curtain in the front opening to minimize the amount of air escaping over the arms of the operator. By using artificial aerosols and a dummy or a test person placing his arms into the working opening of the cabinet, a transfer from the inside to the environment was detected only when the highest concentration of the test aerosol was used. Since the number of bacteria detected was very low, this is considered to be acceptable. When the cabinet was used as a class III type, with a glove panel mounted in the front opening, leakage from the environment occurred. This could be completely prevented by fixing tape over the hinge of the front panel. The conclusion is drawn that this type of biohazard hood can be safely used as a class II and a class III microbiological safety cabinet, provided the construction of the hinge of the front panel will be adapted to prevent transfer from the environment to the working area.

Experimental and clinical gnotobiotics: influence of the microflora on graft-versus-host disease after allogeneic bone marrow transplantation.

One of the major complications of allogeneic bone marrow transplantation (BMT) is graft-versus-host disease (GvHD), which is caused by donor type lymphocytes which react against the recipient's tissues. An important factor which influences GvHD is the recipient's gastrointestinal microflora. This was originally observed in gnotobiotic mice. Infusion of 10(7) H-2 incompatible bone marrow cells into lethally irradiated (9.0 Gy X-rays) conventional mice results in a late onset type GvHD which causes the death of the majority of the recipients during the first two months after BMT. This mortality can be completely prevented if the recipients are germfree mice, or when they are conventional animals which have been subjected to complete or selective gastrointestinal decontamination (GID). In a mouse model, the mechanism responsible for the influence of the microflora on GvHD after allogeneic BMT was investigated. These studies indicate that GvHD can be induced by activated T-lymphocytes from donor origin reacting against bacterial antigens which might be cross-reactive with the recipient's epithelial tissue antigens. Activation of these T-lymphocytes is confined to antigens of certain bacterial species of the recipient which are not present in the indigenous microflora of the donor mice. These bacteria most likely belong to the anaerobic flora of the recipient. The latter hypothesis is strongly supported by the observation in human patients that, in contrast to complete GID, selective decontamination of the gastrointestinal tract did not have any beneficial effect on moderately severe to severe GvHD after transplantation with MHC-matched sibling donor bone marrow grafts.

Selective decontamination of the digestive tract of pregnant rabbits: a method for producing Enterobacteriaceae-free rabbits.

Selective elimination of the Enterobacteriaceae species from the microflora of pregnant rabbits was achieved by the use of nalidixic acid and cotrimoxazole. Animals born under Enterobacteriaceae-free conditions remained so as long as adequate isolation conditions were maintained.

Long-term expression of human adenosine deaminase in rhesus monkeys transplanted with retrovirus-infected bone-marrow cells.

Gene transfer into hemopoietic stem cells could offer a lasting cure for a variety of congenital disorders. As a preclinical test for such a gene therapy, rhesus monkeys were transplanted with autologous bone-marrow cells infected with helper-free recombinant retroviruses carrying the human adenosine deaminase gene. The in vivo regenerative capacity of the infected bone marrow could be conserved, suggesting survival of repopulating hemopoietic stem cells. In the hemopoietic system of transplanted animals the foreign gene could be observed for as long as the animals were analyzed (in two monkeys greater than 1 yr after transplantation). Genetically modified cell types and tissues included peripheral blood mononuclear cells, granulocytes, bone-marrow cells of various densities, and spleen and lymph nodes. The presence of the provirus in the short-living granulocytes greater than 1 yr after bone-marrow transplantation provided evidence for the transduction of very primitive hemopoietic progenitors. Moreover, the gene transfer resulted in sustained production of functional human adenosine deaminase enzyme in peripheral blood mononuclear cells. These results demonstrate the feasibility of bone-marrow gene-therapy approaches, in particular for treating adenosine deaminase deficiency.

Increasing the intestinal resistance of rats to the invasive pathogen Salmonella enteritidis: additive effects of dietary lactulose and calcium.

BACKGROUND AND AIMS: Lactulose fermentation by the intestinal microflora acidifies the gut contents, resulting in an increased resistance to colonisation by acid sensitive pathogens. The extent of fermentation should be controlled to prevent acid induced epithelial cell damage. Considering the buffering capacity of calcium phosphate and its intestinal cytoprotective effects, whether supplemental calcium phosphate adds to the increased resistance to intestinal infections by lactulose fermentations was studied. METHODS: In a strictly controlled experiment, rats were fed a purified low calcium control diet, a low calcium/lactulose diet, or a high calcium/lactulose diet, and subsequently infected orally with Salmonella enteritidis. RESULTS: Lactulose fermentation lowered the pH and increased the lactic acid concentration of the intestinal contents, which significantly reduced excretion of this pathogen in faeces; thus it improved the resistance to colonisation. This agreed with the high sensitivity of S enteritidis to lactic acid (main metabolite of lactulose fermentation) in vitro. Calcium phosphate decreased translocation of S enteritidis to the systemic circulation, an effect independent of lactulose. The unfavourable increased cytotoxicity of faecal water caused by lactulose fermentation was more than counteracted by supplemental calcium phosphate. Moreover, calcium phosphate stimulated lactulose fermentation, as judged by the reduced lactulose excretion in faeces and increased lactic acid, ammonia, and faecal nitrogen excretion. CONCLUSION: Extra calcium phosphate added to a lactulose diet improves the resistance to colonisation and translocation of S enteritidis. This is probably mediated by a calcium induced stimulation of lactulose fermentation by the intestinal microflora and reversion of the lactulose mediated increased luminal cytotoxicity, which reduces damage inflicted on the intestinal mucosa.

The use of a human donor flora for recontamination following antibiotic decontamination.

A method to prevent endogenous infections in patients and laboratory animals with an impaired immune capacity is the total decontamination of the gastrointestinal tract. This is performed by oral administration of nonabsorbable antibiotics. One of the disadvantages of total decontamination is the fact that especially after termination of this treatment the decontaminated individual is easily colonized with organisms from the environment. This is the result of the elimination of the anaerobic part of the microflora, which is responsible for the so-called colonization resistance (CR). This CR is the resistance against exogenous microorganisms to colonize the gastrointestinal tract. In the absence of an anaerobic flora, the colonizing microorganisms can reach abnormally high faecal concentrations, thus increasing the risk for infection. In mice, the implantation of an anaerobic, mouse-derived flora after termination of total decontamination resulted in the restoration of a good CR, as could be shown by orally challenging the animals with a strain of Escherichia coli. Therefore, an anaerobic microflora, free of potentially pathogenic microorganisms, which was isolated from a healthy human volunteer was administered to monkeys and patients after a decontamination period in an attempt to restore CR. In the monkeys, this human donor flora (HDF) did not reduce the faecal concentration of microorganisms that had colonized the gastrointestinal tract before the donor microflora had been established, in contrast to the findings in some of the patients. Qualitative analysis of the microflora of patients which were contaminated with the human donor flora showed that the CR is not of the same quality as is found in healthy individuals. This can be the result of the impaired immune capacity of the patients at the time of HDF implantation. However, the results obtained show that implantation of the HDF in monkeys and patients after a decontamination period allows reconventionalization without an undue risk of microbial infection.

Applicability of solidified water (hydrogel) in laboratory animal care.

Hydrogel was used to provide water in a solid state to rats and mice under laboratory conditions. Hydrogel was easy to handle and readily consumed by the animals. In studies extending over 10 months, no adverse effects on the condition of the animals were observed. Consumption of hydrogel as the sole source of water for nearly 2 months had no effect on the concentration of selected fecal aerobic microorganisms. Histopathological changes were not observed in the tissues of rats and mice kept for periods up to 7 months on hydrogel as the sole source of fluids. The survival time of rats and mice kept on hydrogel after exposure to supralethal doses of total body irradiation did not differ significantly from that of control animals given drinking water in bottles.

Clearence of antibiotics from the intestines after termination of antibiotic decontamination.

The clearance of neomycin and kanamycin from the intestines after stopping oral supply has been determined in mice. Both antibiotics, although given in different doses, were excreted in essentially the same way; the clearance being a little faster than logarithmically in both cases. The importance of this observation with regard to isolation and the moment of reconventionalization is discussed.