Dynamics of the Gut Microbiota in Children Receiving Selective or Total Gut Decontamination Treatment during Hematopoietic Stem Cell Transplantation.

Bloodstream infections and graft-versus-host disease are common complications after hematopoietic stem cell transplantation (HSCT) procedures, associated with the gut microbiota that acts as a reservoir for opportunistic pathogens. Selective gut decontamination (SGD) and total gut decontamination (TGD) during HSCT have been associated with a decreased risk of developing these complications after transplantation. However, because studies have shown conflicting results, the use of these treatments remains subject of debate. In addition, their impact on the gut microbiota is not well studied. The aim of this study was to elucidate the dynamics of the microbiota during and after TGD and to compare these with the dynamics of SGD. In this prospective, observational, single-center study fecal samples were longitudinally collected from 19 children eligible for allogenic HSCT (TGD, n=12; SGD, n=7), weekly during hospital admission and monthly after discharge. In addition, fecal samples were collected from 3 family stem cell donors. Fecal microbiota structure of patients and donors was determined by 16S rRNA gene amplicon sequencing. Microbiota richness and diversity markedly decreased during SGD and TGD and gradually increased after cessation of decontamination treatment. During SGD, gut microbiota composition was relatively stable and dominated by Bacteroides, whereas it showed high inter- and intraindividual variation and low Bacteroides abundance during TGD. In some children TGD allowed the genera Enterococcus and Streptococcus to thrive during treatment. A gut microbiota dominated by Bacteroides was associated with increased predicted activity of several metabolic processes. Comparing the microbiota of recipients and their donors indicated that receiving an SCT did not alter the patient's microbiota to become more similar to that of its donor. Overall, our findings indicate that SGD and TGD affect gut microbiota structure in a treatment-specific manner. Whether these treatments affect clinical outcomes via interference with the gut microbiota needs to be further elucidated.

Variable BCG efficacy in rhesus populations: Pulmonary BCG provides protection where standard intra-dermal vaccination fails.

M.bovis BCG vaccination against tuberculosis (TB) notoriously displays variable protective efficacy in different human populations. In non-human primate studies using rhesus macaques, despite efforts to standardise the model, we have also observed variable efficacy of BCG upon subsequent experimental M. tuberculosis challenge. In the present head-to-head study, we establish that the protective efficacy of standard parenteral BCG immunisation varies among different rhesus cohorts. This provides different dynamic ranges for evaluation of investigational vaccines, opportunities for identifying possible correlates of protective immunity and for determining why parenteral BCG immunisation sometimes fails. We also show that pulmonary mucosal BCG vaccination confers reduced local pathology and improves haematological and immunological parameters post-infection in animals that are not responsive to induction of protection by standard intra-dermal BCG. These results have important implications for pulmonary TB vaccination strategies in the future.

Spontaneous endometriosis in rhesus macaques: evidence for a genetic association with specific Mamu-A1 alleles.

Endometriosis is a poorly understood common debilitating women's reproductive disorder resulting from proliferative and ectopic endometrial tissue associated with variable clinical symptoms including dysmenorrhea (painful menstrual periods), dyspareunia (pain on intercourse), female infertility, and an increased risk of malignant transformation. The rhesus macaque (Macaca mulatta) develops a spontaneous endometriosis that is very similar to that seen in women. We hypothesized that specific major histocompatibility complex (MHC) alleles may contribute to the pathogenesis of endometriosis. As part of a collaboration between the Biomedical Primate Research Centre (BPRC) in the Netherlands and the New England Primate Research Center (NEPRC) in the United States, we analyzed DNA sequences of MHC class I (Macaca mulatta, Mamu-A1) and class II (Mamu-DRB) alleles from rhesus macaques with endometriosis and compared the allele frequencies with those of age-matched healthy macaques. We demonstrate that two MHC class I alleles are overrepresented in diseased macaques compared to controls: Mamu-A1*001, 33.3 % in BPRC animals with endometriosis vs. 11.6 % in healthy macaques ( p =  0.007), and Mamu-A1*007, 21.9 % NEPRC rhesus macaques vs. 6.7 %, ( p =  0.003). We provide evidence that select MHC class I alleles are associated with endometriosis in rhesus macaques and suggest that the disease pathogenesis contribution of MHC class I warrants further research.

History of graft-versus-host disease.

Nuclear warfare at the end of World War II inspired Dick W. van Bekkum to study total-body irradiation (TBI) in animal models. After high-dose TBI, mice died from "primary disease" or bone marrow (BM) aplasia. Intravenous administration of allogeneic BM cells delayed mortality but did not prevent it. Initially the delayed deaths were said to be caused by "secondary disease," which was later renamed graft-versus-host disease (GvHD). GvHD is caused by donor T lymphocytes that destroy recipient cells in skin, intestinal mucosa, bile ducts, and lymph nodes. GvHD is opposed by host-versus-graft disease (HvGD), in which host T lymphocytes destroy the administered allogeneic BM cells, including the administered T lymphocytes of the BM donor. In 1960, van Bekkum became the director of the Radiobiological Institute of the Dutch Organization for Applied Scientific Research TNO, Rijswijk, The Netherlands, where he built a multidisciplinary team that defined the variables controlling the outcome of a BM transplant. The team published their early results in the Journal of Experimental Hematology [1981;9:904-916 and 1956;4:482-488]. Later, protocols were established for BM transplantation (BMT) in patients with severe combined immunodeficiency disease, leukemia, lymphoma, and other diseases of the hematopoietic system. This review honors the scientific contributions made by Dick van Bekkum and his team in defining the four dominant variables for improving the therapeutic ratio of allogeneic BMT and in fostering the international collaboration necessary to translate this knowledge into current clinical practice.

Advantages and Risks of Husbandry and Housing Changes to Improve Animal Wellbeing in a Breeding Colony of Common Marmosets (Callithrix jacchus).

Between 1975 and 2014, housing conditions for laboratory-housed marmosets changed dramatically after the introduction of new guidelines designed to improve their care and wellbeing. According to these guidelines, our facility provided marmosets with outside enclosures, switched to deep litter as bedding material, and discontinued the use of disinfectant agents in animal enclosures. However, both deep litter and access to outside enclosures hypothetically increase the risk of potential exposure to pathogenic microorganisms. We evaluated whether these housing and husbandry modifications constituted an increased veterinary risk for laboratory-housed common marmosets (Callithrix jacchus). After the animals had been exposed to these new housing conditions for 2.5 y, we examined their intestinal bacterial flora and feces, the deep litter, and insects present in the housing. In addition, we assessed the marmosets' general health and the effect of outdoor housing on, for example, vitamin D levels. Although numerous bacterial strains--from nonpathogenic to potentially pathogenic--were cultured, we noted no increase in illness, mortality, or breeding problems related to this environmental microflora. Housing laboratory marmosets in large enriched cages, with both indoor and outdoor enclosures, providing them with deep litter, and eliminating the use of disinfectants present an increased veterinary risk. However, after evaluating all of the collected data, we estimate that the veterinary risk of the new housing conditions is minimal to none in terms of clinical disease, disease outbreaks, abnormal behavior, and negative effects on reproduction.

Complete suppression of the gut microbiome prevents acute graft-versus-host disease following allogeneic bone marrow transplantation.

The hypothesis that elimination of facultative and strict anaerobic microorganisms from the gastro-intestinal tract by antimicrobial drugs in the period of time around allogeneic bone marrow transplantation (BMT) prevents acute graft-versus-host disease (GVHD), was examined in a cohort of 112 children grafted between 1989 and 2002 for hematological malignancies. All patients received T-cell replete marrow from human leukocyte antigens (HLA) matched sibling donors under identical transplantation conditions. To eliminate microorganisms from the gastro-intestinal tract, total gastro-intestinal decontamination (GID) was applied by high doses of non-absorbable antimicrobial drugs while the graft recipient was maintained in strict protective isolation. About half of the children (51%) proved to be successfully decontaminated, and about half (49%) unsuccessfully. One recipient got acute GVHD in the first group and 8 in the second group (p = 0.013). The degree of success of total GID was decisive for the occurrence of acute GVHD, irrespective of the presence of other risk factors such as higher age of recipient and/or donor, female donor for male recipient and carriership or reactivation of herpesviruses. Our results demonstrate that successful total GID of the graft recipient prevents moderate to severe acute GVHD. We suppose that substantial translocation of gastro-intestinal microorganisms or parts of these, functioning as microbial-associated molecular patterns (MAMP's), triggering macrophages/dendritic cells via pattern recognizing receptors (PRR's) is prohibited. As a consequence the initiation and progression of an inflammatory process leading to acute GVHD is inhibited.

Rhesus macaques (Macaca mulatta) are natural hosts of specific Staphylococcus aureus lineages.

Currently, there is no animal model known that mimics natural nasal colonization by Staphylococcus aureus in humans. We investigated whether rhesus macaques are natural nasal carriers of S. aureus. Nasal swabs were taken from 731 macaques. S. aureus isolates were typed by pulsed-field gel electrophoresis (PFGE), spa repeat sequencing and multi-locus sequence typing (MLST), and compared with human strains. Furthermore, the isolates were characterized by several PCRs. Thirty-nine percent of 731 macaques were positive for S. aureus. In general, the macaque S. aureus isolates differed from human strains as they formed separate PFGE clusters, 50% of the isolates were untypeable by agr genotyping, 17 new spa types were identified, which all belonged to new sequence types (STs). Furthermore, 66% of macaque isolates were negative for all superantigen genes. To determine S. aureus nasal colonization, three nasal swabs from 48 duo-housed macaques were taken during a 5 month period. In addition, sera were analyzed for immunoglobulin G and A levels directed against 40 staphylococcal proteins using a bead-based flow cytometry technique. Nineteen percent of the animals were negative for S. aureus, and 17% were three times positive. S. aureus strains were easily exchanged between macaques. The antibody response was less pronounced in macaques compared to humans, and nasal carrier status was not associated with differences in serum anti-staphylococcal antibody levels. In conclusion, rhesus macaques are natural hosts of S. aureus, carrying host-specific lineages. Our data indicate that rhesus macaques are useful as an autologous model for studying S. aureus nasal colonization and infection prevention.

MVA.85A boosting of BCG and an attenuated, phoP deficient M. tuberculosis vaccine both show protective efficacy against tuberculosis in rhesus macaques.

BACKGROUND: Continuous high global tuberculosis (TB) mortality rates and variable vaccine efficacy of Mycobacterium bovis Bacille Calmette-Guérin (BCG) motivate the search for better vaccine regimes. Relevant models are required to downselect the most promising vaccines entering clinical efficacy testing and to identify correlates of protection. METHODS AND FINDINGS: Here, we evaluated immunogenicity and protection against Mycobacterium tuberculosis in rhesus monkeys with two novel strategies: BCG boosted by modified vaccinia virus Ankara expressing antigen 85A (MVA.85A), and attenuated M. tuberculosis with a disrupted phoP gene (SO2) as a single-dose vaccine. Both strategies were well tolerated, and immunogenic as evidenced by induction of specific IFNgamma responses. Antigen 85A-specific IFNgamma secretion was specifically increased by MVA.85A boosting. Importantly, both MVA.85A and SO2 treatment significantly reduced pathology and chest X-ray scores upon infectious challenge with M. tuberculosis Erdman strain. MVA.85A and SO2 treatment also showed reduced average lung bacterial counts (1.0 and 1.2 log respectively, compared with 0.4 log for BCG) and significant protective effect by reduction in C-reactive protein levels, body weight loss, and decrease of erythrocyte-associated hematologic parameters (MCV, MCH, Hb, Ht) as markers of inflammatory infection, all relative to non-vaccinated controls. Lymphocyte stimulation revealed Ag85A-induced IFNgamma levels post-infection as the strongest immunocorrelate for protection (spearman's rho: -0.60). CONCLUSIONS: Both the BCG/MVA.85A prime-boost regime and the novel live attenuated, phoP deficient TB vaccine candidate SO2 showed significant protective efficacy by various parameters in rhesus macaques. Considering the phylogenetic relationship between macaque and man and the similarity in manifestations of TB disease, these data support further development of these primary and combination TB vaccine candidates.

An unusual mode of concerted evolution of the EGF-TM7 receptor chimera EMR2.

The epidermal growth factor (EGF)-TM7 receptors CD97, EMR1, EMR2, EMR3, and EMR4 form a group of adhesion class heptahelical molecules predominantly expressed by cells of the immune system. These receptors bind cellular ligands through EGF-like domains, localized N-terminal to a large extracellular region. Remarkably, EMR2 possesses a chimeric structure with a seven-span transmembrane (TM7) region most related to EMR3 and an EGF domain region nearly identical to CD97. By comparing EGF-TM7 receptors in primates and dogs, we identified an intriguing pattern of concerted evolution, apparently mediated by gene conversion, among EMR2 and the oppositely orientated and physically adjacent genes CD97 and EMR3. This concerted evolution has continuously maintained the chimeric structure of EMR2 since early mammal radiation. Most highly conserved between EMR2 and CD97 is the fourth EGF domain, which mediates binding to chondroitin sulfate, a ligand specificity shared by both receptors. Another ligand, CD55, is bound effectively only by CD97. We show that different molecular mechanisms (mutations vs. alternative splicing) prevent CD55 binding by EMR2 in hominoids. Our findings illustrate how various and partially opposing evolutionary events have shaped the structure and ligand specificity of a modern mammalian gene family.

TB diagnosis in non-human primates: comparison of two interferon-gamma assays and the skin test for identification of Mycobacterium tuberculosis infection.

In general non-human primates are highly susceptible to infections with Mycobacterium tuberculosis which therefore presents an explosive health threat to colonies. To screen for M. tuberculosis infections in non-human primates, the skin test is routinely used. However, the reliability of this test in primates is debatable. The aim of this study was to compare relatively easy in vitro diagnostic tests for TB with the skin test for detection of a tuberculosis (TB) infection. Two in vitro assays, a whole blood interferon-gamma (WB IFN-gamma) assay and in vitro stimulation of isolated lymphocytes (PBMC IFN-gamma) were evaluated during both experimental TB infections in macaques as well as during an outbreak of TB in a macaque quarantine facility. The WB IFN-gamma assay was also evaluated on healthy old and new world monkeys. Our results show that both in vitro assays detected TB infection in macaques. All experimentally infected animals showed TB-specific responses in both assays. In contrast, several TB animals were not diagnosed TB positive using the skin test. In addition, during the outbreak in the quarantine facility one animal was not detected using the routinely used skin test, but it showed strong positive responses in the WB assay. In conclusion, the in vitro assays are a valuable tool for screening non-human primates for TB infection, especially because the assays cause relatively less stress for the animals compared to the skin test and give reproducible and reliable results.