NTP-CERHR expert panel report on the developmental toxicity of soy infant formula

Soy infant formula contains soy protein isolates and is fed to infants as a supplement to or replacement for human milk or cow milk. Soy protein isolates contains estrogenic isoflavones (phytoestrogens) that occur naturally in some legumes, especially soybeans. Phytoestrogens are nonsteroidal, estrogenic compounds. In plants, nearly all phytoestrogens are bound to sugar molecules and these phytoestrogen-sugar complexes are not generally considered hormonally active. Phytoestrogens are found in many food products in addition to soy infant formula, especially soy-based foods such as tofu, soy milk, and in some over-the-counter dietary supplements. Soy infant formula was selected for National Toxicology Program (NTP) evaluation because of (1) the availability of large number of developmental toxicity studies in laboratory animals exposed to the isoflavones found in soy infant formula (namely, genistein) or other soy products, as well as few studies on human infants fed soy infant formula, (2) the availability of information on exposures in infants fed soy infant formula, and (3) public concern for effects on infant or child development. On October 2, 2008 (73 FR 57360), the NTP Center for the Evaluation of Risks to Human Reproduction (CERHR) announced its intention to conduct an updated review of soy infant formula to complete a previous evaluation that was initiated in 2005. Both the current and previous evaluations relied on expert panels to assist the NTP in developing its conclusions on the potential developmental effects associated with the use of soy infant formula, presented in the NTP Brief on Soy Infant Formula. The initial expert panel met on March 15 to 17, 2006, to reach conclusions on the potential developmental and reproductive toxicities of soy infant formula and its predominant isoflavone constituent genistein. The expert panel reports were released for public comment on May 5, 2006 (71 FR 28368). On November 8, 2006 (71 FR 65537), CERHR staff released draft NTP Briefs on Genistein and Soy Formula that provided the NTP's interpretation of the potential for genistein and soy infant formula to cause adverse reproductive and/or developmental effects in exposed humans. However, CERHR did not complete these evaluations, finalize the briefs, or issue NTP Monographs on these substances based on this initial evaluation. Between 2006 and 2009, a substantial number of new publications related to human exposure or reproductive and/or developmental toxicity were published for these substances. Thus, CERHR determined that updated evaluations of genistein and soy infant formula were needed. However, the current evaluation focuses only on soy infant formula and the potential developmental toxicity of its major isoflavone components, e.g. genistein, daidzein (and estrogenic metabolite, equol), and glycitein. This updated evaluation does not include an assessment on the potential reproductive toxicity of genistein following exposures during adulthood as was carried out in the 2006 evaluation. CERHR narrowed the scope of the evaluation because the assessment of reproductive effects of genistein following exposure to adults was not considered relevant to the consideration of soy infant formula use in infants during the 2006 evaluation. To obtain updated information about soy infant formula for the CERHR evaluation, the PubMed (Medline) database was searched from February 2006 to August 2009 with genistein/genistin, daidzein/daidzin, glycitein/glycitin, equol, soy, and other relevant keywords. References were also identified from the bibliographies of published literature. The updated expert panel report represents the efforts of a 14-member panel of government and nongovernment scientists, and was prepared with assistance from NTP staff. The finalized report, released on January 15, 2010 (75 FR 2545), reflects consideration of public comments received on a draft report that was released on October 19, 2009, for public comment and discussions that occurred at a public meeting of the expert panel held December 16 to 18, 2009 (74 FR 53509). The finalized report presents conclusions on (1) the strength of scientific evidence that soy infant formula or its isoflavone constituents are developmental toxicants based on data from in vitro, animal, or human studies; (2) the extent of exposures in infants fed soy infant formula; (3) the assessment of the scientific evidence that adverse developmental health effects may be associated with such exposures; and (4) knowledge gaps that will help establish research and testing priorities to reduce uncertainties and increase confidence in future evaluations. The Expert Panel expressed minimal concern for adverse developmental effects in infants fed soy infant formula. This level of concern represents a "2" on the five-level scale of concern used by the NTP that ranges from negligible concern ("1") to serious concern ("5"). The Expert Panel Report on Soy Infant Formula was considered extensively by NTP staff in preparing the 2010 NTP Brief on Soy Infant Formula, which represents the NTP's opinion on the potential for exposure to soy infant formula to cause adverse developmental effects in humans. The NTP concurred with the expert panel that there is minimal concern for adverse effects on development in infants who consume soy infant formula. This conclusion was based on information about soy infant formula provided in the expert panel report, public comments received during the course of the expert panel evaluation, additional scientific information made available since the expert panel meeting, and peer reviewer critiques of the draft NTP Brief by the NTP Board of Scientific Counselors (BSC) on May 10, 2010 (Meeting materials are available at http://ntp.niehs.nih.gov/go/9741.). The BSC voted in favor of the minimal concern conclusion with 7 yes votes, 3 no votes, and 0 abstentions. One member thought that the conclusion should be negligible concern and two members thought that the level of concern should be higher than minimal concern. The NTP's response to the May 10, 2010 review ("peer-review report") is available on the NTP website at http://ntp.niehs.nih.gov/go/9741. The monograph includes the NTP Brief on Soy Infant Formula as well as the entire final Expert Panel Report on Soy Infant Formula. Public comments received as part of the NTP's evaluation of soy infant formula and other background materials are available at http://cerhr.niehs.nih.gov/evals/index.html.     

Burkholderia cenocepacia BC2L-C is a super lectin with dual specificity and proinflammatory activity

Lectins and adhesins are involved in bacterial adhesion to host tissues and mucus during early steps of infection. We report the characterization of BC2L-C, a soluble lectin from the opportunistic pathogen Burkholderia cenocepacia, which has two distinct domains with unique specificities and biological activities. The N-terminal domain is a novel TNF-α-like fucose-binding lectin, while the C-terminal part is similar to a superfamily of calcium-dependent bacterial lectins. The C-terminal domain displays specificity for mannose and l-glycero-d-manno-heptose. BC2L-C is therefore a superlectin that binds independently to mannose/heptose glycoconjugates and fucosylated human histo-blood group epitopes. The apo form of the C-terminal domain crystallized as a dimer, and calcium and mannose could be docked in the binding site. The whole lectin is hexameric and the overall structure, determined by electron microscopy and small angle X-ray scattering, reveals a flexible arrangement of three mannose/heptose-specific dimers flanked by two fucose-specific TNF-α-like trimers. We propose that BC2L-C binds to the bacterial surface in a mannose/heptose-dependent manner via the C-terminal domain. The TNF-α-like domain triggers IL-8 production in cultured airway epithelial cells in a carbohydrate-independent manner, and is therefore proposed to play a role in the dysregulated proinflammatory response observed in B. cenocepacia lung infections. The unique architecture of this newly recognized superlectin correlates with multiple functions including bacterial cell cross-linking, adhesion to human epithelia, and stimulation of inflammation.     

Effect of low temperature on emergence, fecundity, longevity and host-feeding by Trichogramma brassicae

The occurrence of sub-optimal temperatures during development of immature parasitoids can have important consequences on adult fitness. We investigated the impact of different regimes of low temperature on emergence, differential mortality, longevity and fecundity in Trichogramma brassicae Bezdenko (Hymenoptera: Trichogrammatidae). The host-feeding behaviour of adult females was also measured as an indicator of energy reserve at emergence. Acclimation of 30 days at 10 °C or 24 days at 13 °C allowed T. brassicae immatures to develop with a lower mortality than those exposed directly at 5 °C. Longevity and fecundity of females decreased at a lower rate with acclimation at 10 °C suggesting that acclimation at 13 °C may have depleted the energy reserves of individuals more than acclimation at 10 °C. Short photoperiod exposure during the maternal generation had no effect on progeny’s fitness. We found no difference among the treatments in females’ host-feeding behaviours, in differential mortality at emergence, in female’s mobility and in F1 sex ratio.     

Model for Concomitant Microdialysis Sampling of the Pons and Cerebral Cortex in Rhesus Macaques (Macaca mulatta)

Pediatric diffuse intrinsic pontine gliomas are aggressive brainstem tumors that fail to respond to treatment. We hypothesize that the protective features of the pons may hinder chemotherapeutic agents from entering pontine tissue compared with cortical brain tissue. To test this hypothesis, we developed a unique nonhuman primate model using microdialysis, a continuous in vivo extracellular sampling technique, to compare drug exposure concurrently in pontine tissue, cortical tissue, CSF, and plasma after intravenous administration of chemotherapeutic agents. The surgical coordinates and approach for microdialysis cannula–probe placement were determined in 5 adult male rhesus monkeys (Macaca mulatta) by using MRI. Microdialysis cannulas–probes were implanted stereotactically in the brain, retrodialysis was performed to measure relative recovery, and a 1-h intravenous infusion of temozolomide was administered. Continuous microdialysis samples were collected from the pons and cortex over 4 h with concurrent serial plasma and CSF samples. Postsurgical verification of microdialysis cannula–probe placement was obtained via MRI in 3 macaques and by gross pathology in all 5 animals. The MRI-determined coordinates and surgical methodologies resulted in accurate microdialysis probe placement in the pons and cortex in 4 of the 5 macaques. Histologic examination from these 4 animals revealed negligible tissue damage to the pontine and cortical tissue from microdialysis. One macaque was maintained for 8 wk and had no deficits attributed to the procedure. This animal model allows for the determination of differences in CNS penetration of chemotherapeutic agents in the pons, cortex, and CSF after systemic drug administration.Abbreviations: DIPG, diffuse intrinsic pontine glioma; ECF, extracellular fluidPediatric diffuse intrinsic pontine gliomas (DIPG) are aggressive tumors that cannot be surgically resected due to their location, and are resistant to chemotherapeutic and radiation therapies. As a result, children with DIPG have a dismal prognosis with median survival less than one year from diagnosis. One hypothesis for the poor efficacy of treatment is that innate CNS protective features, such as the blood–brain barrier and the blood–CSF barrier, shield the brainstem to a higher degree given its critical functions, and isolate pontine gliomas from treatment. To test this hypothesis, we developed a nonhuman primate model in rhesus monkeys to evaluate pontine tissue pharmacokinetics by using microdialysis, a continuous in vivo extracellular sampling technique based on diffusion. Microdialysis is the ‘gold standard’ for in vivo sampling methodologies in the CNS, enabling the collection of extracellular tissue fluid via passive diffusion by using a semipermeable membrane probe.A nonhuman primate model demonstrating the feasibility of microdialysis sampling from cortical brain tissue with concurrent pharmacokinetic sampling during chemotherapeutic drug administration has previously been established,^3-5,7 but there are no current animal models that measure drug penetration into the pons. The location of the pons deep within the brain, as well as the vital brainstem functions associated with the pons, present additional obstacles to accurate microdialysis probe placement and sample collection. The objectives of the current study were to develop imaging and surgical procedures for the accurate placement of a microdialysis probe within the pons of rhesus monkeys for sample collection, to establish a method to perform microdialysis simultaneously in multiple CNS regions, and to develop a mechanism to perform repeated microdialysis in the same areas with a single invasive surgical procedure. This model allows for the pharmacokinetic comparison of drug penetration into pontine tissue, in conjunction with cortical tissue, plasma, and CSF, after intravenous administration.     

High Occupancy of Stream Salamanders Despite High Ranavirus Prevalence in a Southern Appalachians Watershed

The interactive effects of environmental stressors and emerging infectious disease pose potential threats to stream salamander communities and their headwater stream ecosystems. To begin assessing these threats, we conducted occupancy surveys and pathogen screening of stream salamanders (Family Plethodontidae) in a protected southern Appalachians watershed in Georgia and North Carolina, USA. Of the 101 salamanders screened for both chytrid fungus (Batrachochytrium dendrobatidis) and Ranavirus, only two exhibited low-level chytrid infections. Prevalence of Ranavirus was much higher (30.4% among five species of Desmognathus). Despite the ubiquity of ranaviral infections, we found high probabilities of site occupancy (≥0.60) for all stream salamander species.     

Application of solid-phase extraction to agar-supported fermentation

Agar-supported fermentation (Ag-SF), a variant of solid-state fermentation, has recently been improved by the development of a dedicated 2 m² scale pilot facility, Platotex. We investigated the application of solid-phase extraction (SPE) to Ag-SF in order to increase yields and minimize the contamination of the extracts with agar constituents. The selection of the appropriate resin was conducted on liquid-state fermentation and Diaion HP-20 exhibited the highest recovery yield and selectivity for the metabolites of the model fungal strains Phomopsis sp. and Fusarium sp. SPE applied to Ag-SF resulted in a particular compartmentalization of the culture. The mycelium that requires oxygen to grow migrates to the top layer and formed a thick biofilm. The resin beads intercalate between the agar surface and the mycelium layer, and trap directly the compounds secreted by the mycelium through a “solid–solid extraction” (SSE) process. The resin/mycelium layer is easily recovered by scraping the surface and the target metabolites extracted by methanol. Ag-SF associated to SSE represents an ideal compromise for the production of bioactive secondary metabolites with limited economic and environmental impact.     

Regulatory CD4+Foxp3+ T Cells Control the Severity of Anaphylaxis

Objective

Anaphylaxis is a life-threatening outcome of immediate-type hypersensitivity to allergen, consecutive to mast cell degranulation by allergen-specific IgE. Regulatory T cells (Treg) can control allergic sensitization and mast cell degranulation, yet their clinical benefit on anaphylactic symptoms is poorly documented. Here we investigated whether Treg action during the effector arm of the allergic response alleviates anaphylaxis.

Methods

We used a validated model of IgE-mediated passive systemic anaphylaxis, induced by intravenous challenge with DNP-HSA in mice passively sensitized with DNP-specific IgE. Anaphylaxis was monitored by the drop in body temperature as well as plasma histamine and serum mMCP1 levels. The role of Treg was analyzed using MHC class II-deficient (Aβ^°/°) mice, treatment with anti-CD25 or anti-CD4 mAbs and conditional ablation of Foxp3⁺ Treg in DEREG mice. Therapeutic efficacy of Treg was also evaluated by transfer experiments using FoxP3-eGFP knock-in mice.

Results

Anaphylaxis did not occur in mast cell-deficient W/W^v mutant mice and was only moderate and transient in mice deficient for histamine receptor-1. Defects in constitutive Treg, either genetic or induced by antibody or toxin treatment resulted in a more severe and/or sustained hypothermia, associated with a rise in serum mMCP1, but not histamine. Adoptive transfer of Foxp3⁺ Treg from either naïve or DNP-sensitized donors similarly alleviated body temperature loss in Treg-deficient DEREG mice.

Conclusion

Constitutive Foxp3⁺ Treg can control the symptomatic phase of mast cell and IgE-dependent anaphylaxis in mice. This might open up new therapeutic avenues using constitutive rather than Ag-specific Treg for inducing tolerance in allergic patients.