The TLR2/TLR6 ligand FSL-1 mitigates radiation-induced hematopoietic injury in mice and nonhuman primates.

Thrombocytopenia, hemorrhage, anemia, and infection are life-threatening issues following accidental or intentional radiation exposure. Since few therapeutics are available, safe and efficacious small molecules to mitigate radiation-induced injury need to be developed. Our previous study showed the synthetic TLR2/TLR6 ligand fibroblast stimulating lipopeptide (FSL-1) prolonged survival and provided MyD88-dependent mitigation of hematopoietic acute radiation syndrome (H-ARS) in mice. Although mice and humans differ in TLR number, expression, and function, nonhuman primate (NHP) TLRs are like those of humans; therefore, studying both animal models is critical for drug development. The objectives of this study were to determine the efficacy of FSL-1 on hematopoietic recovery in small and large animal models subjected to sublethal total body irradiation and investigate its mechanism of action. In mice, we demonstrate a lack of adverse effects, an easy route of delivery (subcutaneous) and efficacy in promoting hematopoietic progenitor cell proliferation by FSL-1. NHP given radiation, followed a day later with a single subcutaneous administration of FSL-1, displayed no adversity but showed elevated hematopoietic cells. Our analyses revealed that FSL-1 promoted red blood cell development and induced soluble effectors following radiation exposure. Cytologic analysis of bone marrow aspirates revealed a striking enhancement of mononuclear progenitor cells in FSL-1-treated NHP. Combining the efficacy of FSL-1 in promoting hematopoietic cell recovery with the lack of adverse effects induced by a single administration supports the application of FSL-1 as a viable countermeasure against H-ARS.

Assessing cerebrovascular reactivity (CVR) in rhesus macaques (Macaca mulatta) using a hypercapnic challenge and pseudo-continuous arterial spin labeling (pCASL).

Cerebrovascular reactivity (CVR) is a measure of cerebral small vessels' ability to respond to changes in metabolic demand and can be quantified using magnetic resonance imaging (MRI) coupled with a vasoactive stimulus. Reduced CVR occurs with neurodegeneration and is associated with cognitive decline. While commonly measured in humans, few studies have evaluated CVR in animal models. Herein, we describe methods to induce hypercapnia in rhesus macaques (Macaca mulatta) under gas anesthesia to measure cerebral blood flow (CBF) and CVR using pseudo-continuous arterial spin labeling (pCASL). Fifteen (13 M, 2 F) adult rhesus macaques underwent pCASL imaging that included a baseline segment (100% O2) followed by a hypercapnic challenge (isoflurane anesthesia with 5% CO2, 95% O2 mixed gas). Relative hypercapnia was defined as an end-tidal CO2 (ETCO2) ≥5 mmHg above baseline ETCO2. The mean ETCO2 during the baseline segment of the pCASL sequence was 34 mmHg (range: 23-48 mmHg). During this segment, mean whole-brain CBF was 51.48 ml/100g/min (range: 21.47-77.23 ml/100g/min). Significant increases (p<0.0001) in ETCO2 were seen upon inspiration of the mixed gas (5% CO2, 95% O2). The mean increase in ETCO2 was 8.5 mmHg and corresponded with a mean increase in CBF of 37.1% (p<0.0001). The mean CVR measured was 4.3%/mmHg. No anesthetic complications occurred as a result of the CO2 challenge. Our methods were effective at inducing a state of relative hypercapnia that corresponds with a detectable increase in whole brain CBF using pCASL MRI. Using these methods, a CO2 challenge can be performed in conjunction with pCASL imaging to evaluate CBF and CVR in rhesus macaques. The measured CVR in rhesus macaques is comparable to human CVR highlighting the translational utility of rhesus macaques in neuroscience research. These methods present a feasible means to measure CVR in comparative models of neurodegeneration and cerebrovascular dysfunction.

Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques.

BACKGROUND: Naturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers. METHODS: We conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation. RESULTS: Similar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs. CONCLUSIONS: These data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC.

Hypertension promotes microbial translocation and dysbiotic shifts in the fecal microbiome of nonhuman primates.

Accumulating evidence indicates a link between gut barrier dysfunction and hypertension. However, it is unclear whether hypertension causes gut barrier dysfunction or vice versa and whether the gut microbiome plays a role. To understand this relationship, we first cross-sectionally examined 153 nonhuman primates [NHPs; Chlorocebus aethiops sabaeus; mean age, 16 ± 0.4 yr; 129 (84.3%) females] for cardiometabolic risk factors and gut barrier function biomarkers. This analysis identified blood pressure and age as specific factors that independently associated with microbial translocation. We then longitudinally tracked male, age-matched spontaneously hypertensive NHPs (Macaca mulatta) to normotensives (n = 16), mean age of 5.8 ± 0.5 yr, to confirm hypertension-related gut barrier dysfunction and to explore the role of microbiome by comparing groups at baseline, 12, and 27 mo. Collectively, hypertensive animals in both studies showed evidence of gut barrier dysfunction (i.e., microbial translocation), as indicated by higher plasma levels of lipopolysaccharide-binding protein (LBP)-1, when compared with normotensive animals. Furthermore, plasma LBP-1 levels were correlated with diastolic blood pressure, independent of age and other health markers, suggesting specificity of the effect of hypertension on microbial translocation. In over 2 yr of longitudinal assessment, hypertensive animals had escalating plasma levels of LBP-1 and greater bacterial gene expression in mesenteric lymph nodes compared with normotensive animals, confirming microbes translocated across the intestinal barrier. Concomitantly, we identified distinct shifts in the gut microbial signature of hypertensive versus normotensive animals at 12 and 27 mo. These results suggest that hypertension contributes to microbial translocation in the gut and eventually unhealthy shifts in the gut microbiome, possibly contributing to poor health outcomes, providing further impetus for the management of hypertension.NEW & NOTEWORTHY Hypertension specifically had detrimental effects on microbial translocation when age and metabolic syndrome criteria were evaluated as drivers of cardiovascular disease in a relevant nonhuman primate model. Intestinal barrier function exponentially decayed over time with chronic hypertension, and microbial translocation was confirmed by detection of more microbial genes in regional draining lymph nodes. Chronic hypertension resulted in fecal microbial dysbiosis and elevations of the biomarker NT-proBNP. This study provides insights on the barrier dysfunction, dysbiosis, and hypertension in controlled studies of nonhuman primates. Our study includes a longitudinal component comparing naturally occurring hypertensive to normotensive primates to confirm microbial translocation and dysbiotic microbiome development. Hypertension is an underappreciated driver of subclinical endotoxemia that can drive chronic inflammatory diseases.

Organ Weights in Relation to Age and Sex in Cynomolgus Monkeys (Macaca fascicularis).

Laboratory animal research is an important contributor to both human and animal medicine. Currently, there is extensive use of cynomolgus monkeys (Macaca fascicularis) in pathology and toxicology research. The purpose of this study was to define reference values for absolute and percentage organ weights in M fascicularis of different ages and sex. Organ weights were obtained from necropsies of 1022 cynomolgus monkeys at the Wake Forest School of Medicine from 1997 to 2018. Distributions of absolute and percentage weights for each organ were described; sex and age groups were compared using analysis of variance. Age effects on percentage of body weights for each organ were analyzed within each sex. Diet effects were also analyzed. This evaluation showed that male body weights and absolute organ weights were greater for all age groups; however, female organ to body weight percentages were greater for most organs. Percentage of organ weight to body weight declined for the adrenals, brain, lung, thyroid and thymus during maturation, whereas percentage weight of pancreas, prostate, testes, and uterus increased. Animals consuming a high-fat, Western-type diet had a lower body weight than animals consuming a carbohydrate-rich chow diet. This information will be useful for further toxicology and pathology studies concerning cynomolgus monkeys.

Investigating the role of endogenous estrogens, hormone replacement therapy, and blockade of estrogen receptor-α activity on breast metabolic signaling.

PURPOSE: Menopause is associated with an increased risk of estrogen receptor-positive (ER +) breast cancer. To characterize the metabolic shifts associated with reduced estrogen bioavailability on breast tissue, metabolomics was performed from ovary-intact and ovariectomized (OVX) female non-human primates (NHP). The effects of exogenous estrogen administration or estrogen receptor blockade (tamoxifen treatment) on menopause-induced metabolic changes were also investigated. METHODS: Bilateral ovariectomies were performed on female cynomolgus macaques (Macaca fascicularis) to model menopause. OVX NHP were then divided into untreated (n = 13), conjugated equine estrogen (CEE)-treated (n= 13), or tamoxifen-treated (n = 13) subgroups and followed for 3 years. Aged-matched ovary-intact female NHP (n = 12) were used as a premenopausal comparison group. Metabolomics was performed on snap-frozen breast tissue. RESULTS: Changes in several different metabolic biochemicals were noted, particularly in glucose and fatty acid metabolism. Specifically, glycolytic, Krebs cycle, acylcarnitines, and phospholipid metabolites were elevated in breast tissue from ovary-intact NHP and OVX + CEE in relation to the OVX and OVX + tamoxifen group. In contrast, treatment with CEE and tamoxifen decreased several cholesterol metabolites, compared to the ovary-intact and OVX NHP. These changes were accompanied by elevated bile acid metabolites in the ovary-intact group. CONCLUSION: Alterations in estrogen bioavailability are associated with changes in the mammary tissue metabolome, particularly in glucose and fatty acid metabolism. Changes in these pathways may represent a bioenergetic shift in gland metabolism at menopause that may affect breast cancer risk.

IL-1ß Stimulates Brain-Derived Neurotrophic Factor Production in Eutopic Endometriosis Stromal Cell Cultures: A Model for Cytokine Regulation of Neuroangiogenesis.

Endometriosis implants are comprised of glandular and stromal elements, macrophages, nerves, and blood vessels and are commonly accompanied by pelvic pain. We propose that activated macrophages are recruited to and infiltrate nascent lesions, where they secrete proinflammatory cytokines, promoting the production of chemokines, neurotrophins, and angiogenic growth factors that sustain an inflammatory microenvironment. Immunohistochemical evaluation of endometriosis lesions reveals in situ colocalization of concentrated macrophages, brain-derived neurotrophic factor (BDNF), and nerve fibers. These observations were coupled with biochemical analyses of primary eutopic endometriosis stromal cell (EESC) cultures, which allowed defining potential pathways leading to the neuroangiogenic phenotype of these lesions. Our findings indicate that IL-1ß potently (EC50 = 7 ± 2 ng/mL) stimulates production of EESC BDNF at the mRNA and protein levels in an IL-1 receptor-dependent fashion. Selective kinase inhibitors demonstrate that this IL-1ß effect is mediated by c-Jun N-terminal kinase (JNK), NF-κB, and mechanistic target of rapamycin signal transduction pathways. IL-1ß regulation of regulated on activation normal T cell expressed and secreted (RANTES), a prominent EESC chemokine, also relies on JNK and NF-κB. An important clinical implication of the study is that interference with BDNF and RANTES production, by selectively targeting the JNK and NF-κB cascades, may offer a tractable therapeutic strategy to mitigate the pain and inflammation associated with endometriosis.

Endocrine Disruption and Reproductive Pathology.

During the past 20 years, investigations involving endocrine active substances (EAS) and reproductive toxicity have dominated the landscape of ecotoxicological research. This has occurred in concert with heightened awareness in the scientific community, general public, and governmental entities of the potential consequences of chemical perturbation in humans and wildlife. The exponential growth of experimentation in this field is fueled by our expanding knowledge into the complex nature of endocrine systems and the intricacy of their interactions with xenobiotic agents. Complicating factors include the ever-increasing number of novel receptors and alternate mechanistic pathways that have come to light, effects of chemical mixtures in the environment versus those of single EAS laboratory exposures, the challenge of differentiating endocrine disruption from direct cytotoxicity, and the potential for transgenerational effects. Although initially concerned with EAS effects chiefly in the thyroid glands and reproductive organs, it is now recognized that anthropomorphic substances may also adversely affect the nervous and immune systems via hormonal mechanisms and play substantial roles in metabolic diseases, such as type 2 diabetes and obesity.

Altered expression of glial markers, chemokines, and opioid receptors in the spinal cord of type 2 diabetic monkeys.

Neuroinflammation is a pathological condition that underlies diabetes and affects sensory processing. Given the high prevalence of pain in diabetic patients and crosstalk between chemokines and opioids, it is pivotal to know whether neuroinflammation-associated mediators are dysregulated in the central nervous system of diabetic primates. Therefore, the aim of this study was to investigate whether mRNA expression levels of glial markers, chemokines, and opioid receptors are altered in the spinal cord and thalamus of naturally occurring type 2 diabetic monkeys (n=7) compared with age-matched non-diabetic monkeys (n=6). By using RT-qPCR, we found that mRNA expression levels of both GFAP and IBA1 were up-regulated in the spinal dorsal horn (SDH) of diabetic monkeys compared with non-diabetic monkeys. Among all chemokines, expression levels of three chemokine ligand-receptor systems, i.e., CCL2-CCR2, CCL3-CCR1/5, and CCL4-CCR5, were up-regulated in the SDH of diabetic monkeys. Moreover, in the SDH, seven additional chemokine receptors, i.e., CCR4, CCR6, CCR8, CCR10, CXCR3, CXCR5, and CXCR6, were also up-regulated in diabetic monkeys. In contrast, expression levels of MOP, KOP, and DOP, but not NOP receptors, were down-regulated in the SDH of diabetic monkeys, and the thalamus had fewer changes in the glial markers, chemokines and opioids. These findings indicate that neuroinflammation, manifested as glial activation and simultaneous up-regulation of multiple chemokine ligands and receptors, seems to be permanent in type 2 diabetic monkeys. As chemokines and opioids are important pain modulators, this first-in-primate study provides a translational bridge for determining the functional efficacy of spinal drugs targeting their signaling cascades.

DMP1ß, a splice isoform of the tumour suppressor DMP1 locus, induces proliferation and progression of breast cancer.

Our recent work has indicated that the DMP1 locus on 7q21, encoding a haplo-insufficient tumour suppressor, is hemizygously deleted at a high frequency in breast cancer. The locus encodes DMP1α protein, an activator of the p53 pathway leading to cell cycle arrest and senescence, and two other functionally undefined isoforms, DMP1ß and DMP1γ. In this study, we show that the DMP1 locus is alternatively spliced in ∼30% of breast cancer cases with relatively decreased DMP1α and increased DMP1ß expression. RNA-seq analyses of a publicly available database showed significantly increased DMP1ß mRNA in 43-55% of human breast cancers, dependent on histological subtypes. Similarly, DMP1ß protein was found to be overexpressed in ∼60% of tumours relative to their surrounding normal tissue. Importantly, alteration of DMP1 splicing and DMP1ß overexpression were associated with poor clinical outcomes of the breast cancer patients, indicating that DMP1ß may have a biological function. Indeed, DMP1ß increased proliferation of non-tumourigenic mammary epithelial cells and knockdown of endogenous DMP1 inhibited breast cancer cell growth. To determine DMP1ß's role in vivo, we established MMTV-DMP1ß transgenic mouse lines. DMP1ß overexpression was sufficient to induce mammary gland hyperplasia and multifocal tumour lesions in mice at 7-18 months of age. The tumours formed were adenosquamous carcinomas with evidence of transdifferentiation and keratinized deposits. Overall, we identify alternative splicing as a mechanism utilized by cancer cells to modulate the DMP1 locus through diminishing DMP1α tumour suppressor expression, while simultaneously up-regulating the tumour-promoting DMP1ß isoform.

Novel In Vivo model for combinatorial fluorescence labeling in mouse prostate.

BACKGROUND: The epithelial layer of prostate glands contains several types of cells, including luminal and basal cells. Yet there is paucity of animal models to study the cellular origin of normal or neoplastic development in the prostate to facilitate the treatment of heterogenous prostate diseases by targeting individual cell lineages. METHODS: We developed a mouse model that expresses different types of fluorescent proteins (XFPs) specifically in prostatic cells. Using an in vivo stochastic fluorescent protein combinatorial strategy, XFP signals were expressed specifically in prostate of Protein Kinase D1 (PKD1) knock-out, K-Ras(G) (12) (D) knock-in, and Phosphatase and tensin homolog (PTEN) and PKD1 double knock-out mice under the control of PB-Cre promoter. RESULTS: In vivo XFP signals were observed in prostate of PKD1 knock-out, K-Ras(G) (12) (D) knock-in, and PTEN PKD1 double knock-out mice, which developed normal, hyperplastic, and neoplastic prostate, respectively. The patchy expression pattern of XFPs in neoplasia tissue indicated the clonal origin of cancer cells in the prostate. CONCLUSIONS: The transgenic mouse models demonstrate combinatorial fluorescent protein expression in normal and cancerous prostatic tissues. This novel prostate-specific fluorescent labeled mouse model, which we named Prorainbow, could be useful in studying benign and malignant pathology of prostate.

Hormone Receptor Expression in Spontaneous Uterine Adenocarcinoma in Fischer 344 Rats.

Most uterine cancers, the most common gynecological malignancies in women in developed countries, are hormone-dependent endometrial adenocarcinomas (EACs) that express estrogen and progesterone receptors. Although rat strains exist with a high spontaneous incidence of EAC, the Fischer 344 (F344) strain, previously one of the most commonly used strains in carcinogenicity testing, is not a high-incidence strain. To better understand the biology of this neoplasm, we assessed estrogen receptor α (ER), progesterone receptor (PR), and Ki-67 expression using immunohistochemistry in spontaneous EAC in 18 F344 rats used as control animals in 2-year National Toxicology Program bioassays. Of the 18 tumors, 9 were well-differentiated and 9 were poorly differentiated. Most tumors, 7/18, were ER+PR+, as observed in women. Of the remainder, 6/18 were ER+PR-, 2/18 were ER-PR+, and 3/18 were ER-PR-. Well-differentiated tumors were ER+ (8/9) more often than poorly differentiated tumors (5/9). The percentage of ER+ tumors (72%) in rats was similar to that seen in women, but rats less frequently had PR+ (50%) tumors than women. The heterogeneous estrogen and progesterone receptor immunophenotypes observed in F344 rats in this study highlight the importance of evaluating hormone receptor expression in animal models used for chemical evaluations.

Assessment of Multiplate platelet aggregometry using citrate, heparin or hirudin in Rhesus macaques.

Electrical impedance aggregometry (EIA) has gained popularity in clinical and research applications. Nonhuman primates are used to study disease and drug-related mechanisms that affect hemostasis, therefore establishing normal EIA parameters are necessary. The anticoagulants sodium heparin, hirudin and sodium citrate and three agonists, ADP, ASPI, and collagen were evaluated. Whole blood from 12 adult male rhesus macaques was collected to evaluate anticoagulants, sodium heparin, hirudin and sodium citrate using three agonists (ADP, ASPI and collagen), on the Multiplate® 5.0 Analyzer. Platelet function was reported for three parameters: Area under the curve (AUC), aggregation, and aggregation velocity. There was a significant difference in mean AUC between citrate and heparin samples, and citrate and hirudin samples regardless of the agonist used. There was no difference in AUC between heparin and hirudin. ADP-activated samples showed an increase in impedance with hirudin samples compared to citrate. Furthermore, heparin and hirudin out-perform citrate as the anticoagulant for EIA in the macaque. Finally, this study demonstrates the utility of the Multiplate® system in this model and provides important insight into anticoagulant choice when using EIA.

Tamoxifen-DNA adduct formation in monkey and human reproductive organs.

The estrogen analog tamoxifen (TAM), used for adjuvant therapy of breast cancer, induces endometrial and uterine tumors in breast cancer patients. Proliferation stimulus of the uterine endometrium is likely involved in tumor induction, but genotoxicity may also play a role. Formation of TAM-DNA adducts in human tissues has been reported but remains controversial. To address this issue, we examined TAM-DNA adducts in uteri from two species of monkeys, Erythrocebus patas (patas) and Macaca fascicularis (macaque), and in human endometrium and myometrium. Monkeys were given 3-4 months of chronic TAM dosing scaled to be equivalent to the daily human dose. In the uteri, livers and brains from the patas (n = 3), and endometrium from the macaques (n = 4), TAM-DNA adducts were measurable by TAM-DNA chemiluminescence immunoassay. Average TAM-DNA adduct values for the patas uteri (23 adducts/10(8) nucleotides) were similar to those found in endometrium of the macaques (19 adducts/10(8) nucleotides). Endometrium of macaques exposed to both TAM and low-dose estradiol (n = 5) averaged 34 adducts/10(8) nucleotides. To examine TAM-DNA persistence in the patas, females (n = 3) were exposed to TAM for 3 months and to no drug for an additional month, resulting in low or non-detectable TAM-DNA in livers and uteri. Human endometrial and myometrial samples from women receiving (n = 8) and not receiving (n = 8) TAM therapy were also evaluated. Women receiving TAM therapy averaged 10.3 TAM-DNA adducts/10(8) nucleotides, whereas unexposed women showed no detectable TAM-DNA. The data indicate that genotoxicity, in addition to estrogen agonist effects, may contribute to TAM-induced human endometrial cancer.

Targeted deletion of RasGRP1 impairs skin tumorigenesis.

Ras is frequently activated in cutaneous squamous cell carcinoma, a prevalent form of skin cancer. However, the pathways that contribute to Ras-induced transformation have not been entirely elucidated. We have previously demonstrated that in transgenic mice, overexpression of the Ras activator RasGRP1 promotes the formation of spontaneous skin tumors and enhances malignant progression in the multistage carcinogenesis skin model that relies on the oncogenic activation of H-Ras. Utilizing a RasGRP1 knockout mouse model (RasGRP1 KO), we now show that lack of RasGRP1 reduced the susceptibility to skin tumorigenesis. The dependency on RasGRP1 was associated with a diminished response to the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Specifically, we found impairment of epidermal hyperplasia induced by TPA through keratinocyte proliferation. Using a keratinocyte cell line that carries a ras oncogenic mutation, we also demonstrated that RasGRP1 could further activate Ras in response to TPA. Thus, we propose that RasGRP1 upregulates signaling from Ras and contributes to epidermal tumorigenesis by increasing the total dosage of active Ras.

The placenta in toxicology. Part III: Pathologic assessment of the placenta.

This short review is derived from the peer-reviewed literature and the experience and case materials of the authors. Brief illustrated summaries are presented on the gross and histologic normal anatomy of rodent and macaque placentas, including typical organ weights, with comments on differences from the human placenta. Common incidental findings, background lesions, and induced toxic lesions are addressed, and a recommended strategy for pathologic evaluation of placentas is provided.

The placenta in toxicology. Part IV: Battery of toxicological test systems based on human placenta.

This review summarizes the potential and also some limitations of using human placentas, or placental cells and structures for toxicology testing. The placenta contains a wide spectrum of cell types and tissues, such as trophoblast cells, immune cells, fibroblasts, stem cells, endothelial cells, vessels, glands, membranes, and many others. It may be expected that in many cases the relevance of results obtained from human placenta will be higher than those from animal models due to species specificity of metabolism and placental structure. For practical and economical reasons, we propose to apply a battery of sequential experiments for analysis of potential toxicants. This should start with using cell lines, followed by testing placenta tissue explants and isolated placenta cells, and finally by application of single and dual side ex vivo placenta perfusion. With each of these steps, the relative workload increases while the number of feasible repeats decreases. Simultaneously, the predictive power enhances by increasing similarity with in vivo human conditions. Toxic effects may be detected by performing proliferation, vitality and cell death assays, analysis of protein and hormone expression, immunohistochemistry or testing functionality of signaling pathways, gene expression, transport mechanisms, and so on. When toxic effects appear at any step, the subsequent assays may be cancelled. Such a system may be useful to reduce costs and increase specificity in testing questionable toxicants. Nonetheless, it requires further standardization and end point definitions for better comparability of results from different toxicants and to estimate the respective in vivo translatability and predictive value.

The placenta in toxicology. Part I: Animal models in toxicology: placental morphology and tolerance molecules in the cynomolgus monkey (Macaca fascicularis).

The immune system represents a key defense mechanism against potential pathogens and adverse non-self materials. During pregnancy, the placenta is the point of contact between the maternal organism and non-self proteins of the fetal allograft and hence undoubtedly fulfils immune functions. In the placenta bacteria, foreign (non-self) proteins and proteins that might be introduced in toxicological studies or by medication are barred from reaching the progeny, and the maternal immune system is primed for acceptance of non-maternal fetal protein. Both immunologic protection of the fetus and acceptance of the fetus by the mother require effective mechanisms to prevent an immunologic fetomaternal conflict and to keep both organisms in balance. This is why the placenta requires toxicological consideration in view of its immune organ function. The following articles deal with placenta immune-, control-, and tolerance mechanisms in view of both fetal and maternal aspects. Furthermore, models for experimental access to placental immune function are addressed and the pathological evaluation is elucidated. "The Placenta as an Immune Organ and Its Relevance in Toxicological Studies" was subject of a continuing education course at the 2012 Society of Toxicologic Pathology meeting held in Boston, MA.

The placenta in toxicology. Part II: Systemic and local immune adaptations in pregnancy.

During pregnancy, the maternal immune system is challenged by the semiallogeneic fetus, which must be tolerated without compromising fetal or maternal health. This review updates the systemic and local immune changes taking place during human pregnancy, including some examples in rodents. Systemic changes are induced by contact of maternal blood with placental factors and include enhanced innate immunity with increased activation of granulocytes and nonclassical monocytes. Although a bias toward T helper (Th2) and regulatory T cell (Treg) immunity has been associated with healthy pregnancy, the relationship between different circulating Th cell subsets is not straightforward. Instead, these adaptations appear most evidently at the fetal-maternal interface, where for instance Tregs are enriched and promote fetal tolerance. Also innate immune cells, that is, natural killer cells and macrophages, are enriched, constituting the majority of decidual leukocytes. These cells not only contribute to immune regulation but also aid in establishing the placenta by promoting trophoblast recruitment and angiogenesis. Thus, proper interaction between leukocytes and placental trophoblasts is necessary for normal placentation and immune adaptation. Consequently, spontaneous maladaptation or interference of the immune system with toxic substances may be important contributing factors for the development of pregnancy complications such as preeclampsia, preterm labor, and recurrent miscarriages.

Role of ERα and Aromatase in Juvenile Gigantomastia.

CONTEXT: Approximately 150 patients with juvenile gigantomastia have been reported in the literature but the underlying biologic mechanisms remain unknown. OBJECTIVE: To conduct extensive clinical, biochemical, immunochemical, and genetic studies in 3 patients with juvenile gigantomastia to determine causative biologic factors. METHODS: We examined clinical effects of estrogen by blockading estrogen synthesis or its action. Breast tissue aromatase expression and activity were quantitated in 1 patient and 5 controls. Other biochemical markers, including estrogen receptor α (ERα), cyclin D1 and E, p-RB, p-MAPK, p-AKT, BCL-2, EGF-R, IGF-IR ß, and p-EGFR were assayed by Western blot. Immunohistochemical analyses for aromatase, ERα and ß, PgR, Ki67, sulfotransferase, estrone sulfatase, and 17ßHD were performed in all 3 patients. The entire genomes of the mother, father, and patient in the 3 families were sequenced. RESULTS: Blockade of estrogen synthesis or action in patients resulted in demonstrable clinical effects. Biochemical studies on fresh frozen tissue revealed no differences between patients and controls, presumably due to tissue dilution from the large proportion of stroma. However, immunohistochemical analysis of ductal breast cells in the 3 patients revealed a high percent of ERα (64.1% ± 7.8% vs reference women 9.6%, range 2.3-15%); aromatase score of 4 (76%-100% of cells positive vs 30.4% ± 5.6%); PgR (69.5% ± 15.2% vs 6.0%, range 2.7%-11.9%) and Ki67 (23.7% ± 0.54% vs 4.2%). Genetic studies were inconclusive although some intriguing variants were identified. CONCLUSION: The data implicate an important biologic role for ERα to increase tissue sensitivity to estrogen and aromatase to enhance local tissue production as biologic factors involved in juvenile gigantomastia.