Assessing the Safety of Mechanically Fibrillated Cellulose Nanofibers (fib-CNF) via Toxicity Tests on Mice: Single Intratracheal Administration and 28 Days' Oral Intake.

Mechanically fibrillated cellulose nanofibers, known as fib-CNF (fiber length: 500 nm; diameter: 45 nm), are used in composites and as a natural thickener in foods. To evaluate their safety, we conducted a 28-day study in mice with inhalation exposure at 0.2 mg/body and oral administration of 400 mg/kg/day. Inhalation exposure to fib-CNF caused transient weight loss, changes in blood cell counts, and increased lung weights. These changes were attributed to adaptive responses. The oral administration of fib-CNF for 28 days resulted in no apparent toxic effects except for a slight decrease in platelet counts. The fib-CNF administration using the protocols studied appears to be safe in mice.

NELF and PAF1C complexes are core transcriptional machineries controlling colon cancer stemness.

Mutations in APC, found in 80% of colon caner, enhance ß-catenin stabilization, which is the initial step of colonic tumorigenesis. However, the core transcriptional mechanism underlying the induction of colon cancer stemness by stable ß-catenin remains unclear. Here, we found that inducible inhibition of ß-catenin suppressed elongation of Pol II and RNA polymerase-associated factor 1 complex (PAF1C) around the transcription start site (TSS) of LGR5. Moreover, stable ß-catenin enhanced the formation of active Pol II complex cooperatively with CDC73 and CDK9 by facilitating the recruitment of DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) complexes to the Pol II complex. Subsequently, stable ß-catenin facilitated the formation of the Pol II-DSIF-PAF1C complex, suggesting that stable ß-catenin induces cancer stemness by stimulating active Pol II complex through NELF and PAF1C. Furthermore, NELF or PAF1C inhibition recapitulated the changes in cancer stemness-related gene expression induced by the inhibition of stable ß-catenin and suppressed colon cancer stemness. Additionally, the chemical inhibition of CDK12 (a downstream transcription CDK of PAF1C) suppressed colon cancer stemness. These results suggest that NELF and PAF1C are the core transcriptional machineries that control expression of colon cancer stemness-inducing genes and may be therapeutic targets for colon cancer.

Successful conservative management in a dog with substantial urinary bladder ischemia.

A 7-year-old spayed female pug dog was brought to the veterinary college with a severely and diffusely ischemic urinary bladder secondary to obstructive uroliths in the lower urinary tract. Cystotomy was performed to remove the uroliths and the ischemic bladder was managed with conservative treatment. A recheck abdominal ultrasound 4.5 mo after surgery revealed an abdominal mass that was associated with the urinary bladder. An exploratory laparotomy and partial cystectomy were performed. Histopathology of the mass showed granulomatous inflammation centered on necrotic tissue. The dog recovered well, and long-term prognosis is good. To the best of our knowledge, this is the first veterinary case report describing conservative management of an ischemic urinary bladder. An uncommon complication following cystotomy and the relevant imaging findings is also described. The positive outcome for the dog demonstrated that conservative management may serve as an option for treatment of substantial ischemia of the urinary bladder.

Prise en charge conservatrice réussie chez un chien présentant une ischémie importante de la vessie. Une chienne carlin femelle stérilisée âgée de 7 ans a été amenée à l'école vétérinaire avec une vessie sévèrement et diffusément ischémique secondaire à des urolithes obstructifs dans les voies urinaires inférieures. Une cystotomie a été réalisée pour retirer les urolithes et la vessie ischémique a été prise en charge avec un traitement conservateur. Une échographie abdominale de contrôle 4,5 mois après la chirurgie a révélé une masse abdominale associée à la vessie. Une laparotomie exploratrice et une cystectomie partielle ont été réalisées. L'histopathologie de la masse a montré une inflammation granulomateuse centrée sur le tissu nécrotique. Le chien a bien récupéré et le pronostic à long terme est bon. À notre connaissance, il s'agit du premier rapport de cas vétérinaire décrivant la prise en charge conservatrice d'une vessie ischémique. Une complication peu fréquente après cystotomie et les résultats d'imagerie pertinents sont également décrits. Le résultat positif pour le chien a démontré qu'une gestion conservatrice peut servir d'option pour le traitement d'une ischémie importante de la vessie.(Traduit par Dr Serge Messier).

Self-reported radiation safety behaviors among veterinary specialists and residents performing fluoroscopic procedures on small animals.

OBJECTIVE: To describe the radiation safety behaviors of veterinary specialists performing small animal fluoroscopic procedures and examine potential risk factors for these behaviors, including knowledge of radiation risk and training regarding machine operating parameters. SAMPLE: 197 veterinary specialists and residents in training. PROCEDURES: An electronic questionnaire was distributed to members of the American Colleges of Veterinary Internal Medicine (subspecialties of cardiology and small animal internal medicine), Veterinary Radiology, and Veterinary Surgery. RESULTS: The overall survey response rate was 6% (240/4,274 email recipients). Of the 240 respondents, 197 (82%) had operated an x-ray unit for a small animal fluoroscopic procedure in the preceding year and fully completed the questionnaire. More than 95% of respondents believed that radiation causes cancer, yet approximately 60% of respondents never wore hand or eye protection during fluoroscopic procedures, and 28% never adjusted the fluoroscopy machine operating parameters for the purpose of reducing their radiation dose. The most common reasons for not wearing eye shielding included no requirement to wear eyeglasses, poor fit, discomfort, and interference of eyeglasses with task performance. Respondents who had received training regarding machine operating parameters adjusted those parameters to reduce their radiation dose during procedures significantly more frequently than did respondents who had not received training. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of the self-reported suboptimal radiation safety practices among veterinary fluoroscopy users, we recommend formal incorporation of radiation safety education into residency training programs. All fluoroscopy machine operators should be trained regarding the machine operating parameters that can be adjusted to reduce occupational radiation exposure.

Effect of irrigation technique on the vertebral canal temperature during thoracolumbar hemilaminectomy in dogs: An ex vivo study.

OBJECTIVE: To determine the influence of continuous and intermittent bolus irrigation on vertebral canal temperature during thoracolumbar hemilaminectomy. STUDY DESIGN: Ex vivo study. SAMPLE POPULATION: Ten canine cadavers. METHODS: Six consecutive thoracolumbar hemilaminectomies starting at T12-13 with alternating left- or right-side selection, and alternating continuous or intermittent bolus irrigation were performed in each dog resulting in 30 hemilaminectomies per irrigation technique. Drilling was performed for 15 s followed by a 10-s pause and resumed until completion of hemilaminectomy. Continuous irrigation consisted of saline delivered at 15 ml/min during drilling. Bolus irrigation consisted of manual delivery of 10 ml saline during the pause. Temperatures were recorded with two sensors placed within the vertebral canal adjacent to target hemilaminectomy site and compared between techniques with a linear mixed model. RESULTS: Intermittent bolus irrigation was associated with lower peak vertebral canal temperatures (mean 15.7°C; range 9.4-23.3°C) than continuous irrigation (mean 16.7°C; range 9.6-27.6°C, p = .003) (mean difference of 1.1°C, p = .006). Similarly, mean vertebral canal temperatures remained lower when hemilaminectomies were performed under intermittent rather than continuous irrigation (mean difference of 0.48°C, p = .006, linear mixed model). CONCLUSION: Lower vertebral canal temperatures were maintained during hemilaminectomy with intermittent bolus rather than continuous irrigation. CLINICAL SIGNIFICANCE: Both intermittent bolus and continuous irrigation are suitable to prevent elevations in canine vertebral canal temperature during hemilaminectomy.

A multi-organ infection involving a left adrenal gland abscess in a diabetic dog.

A 14-year-old, intact male, American cocker spaniel dog was presented with a 5-day history of lethargy, inappetence, loose stools, abdominal pain, and hematuria. The dog had a 4-year history of diabetes mellitus and was in remission for immune-mediated hemolytic anemia. Abdominal ultrasonography and computed tomography scan revealed diffuse left adrenomegaly, splenic nodules, prostatomegaly, bilateral testicular cysts, and a hepatic mass. The dog underwent an exploratory laparotomy and a left adrenalectomy. Histopathology of the left adrenal gland was consistent with an adrenal abscess. To the authors' knowledge, this is the first reported case of an adrenal gland abscess in a dog. Key clinical message: This case highlights the potential for multi-organ infections in uncontrolled diabetic patients with non-specific clinical signs.

Une infection multi-organes impliquant un abcès de la glande surrénale gauche chez un chien diabétique. Un cocker américain mâle de 14 ans, intact, a été présenté avec de la léthargie, de l'inappétence, des selles molles, des douleurs abdominales et de l'hématurie depuis 5 jours. Le chien avait une histoire de 4 ans de diabète sucré et était en rémission pour une anémie hémolytique à médiation immunitaire. L'échographie abdominale et la tomodensitométrie ont révélé une adrénomégalie diffuse gauche, des nodules spléniques, une prostatomégalie, des kystes testiculaires bilatéraux et une masse hépatique. Le chien a subi une laparotomie exploratoire et une ablation de la surrénale gauche. L'histopathologie de la glande surrénale gauche était compatible avec un abcès surrénalien. À la connaissance des auteurs, il s'agit du premier cas signalé d'abcès des glandes surrénales chez un chien.Message clinique cléCe cas met en évidence le potentiel d'infections multi-organes chez les patients diabétiques non maitrisés présentant des signes cliniques non spécifiques.(Traduit par Dr Serge Messier).

Effects of shoulder distraction on canine shoulder arthroscopy.

OBJECTIVE: To evaluate the use of the Leipzig distractor during canine shoulder arthroscopy. STUDY DESIGN: Experimental, ex vivo. SAMPLE POPULATION: Paired shoulder joints from 15 large breed canine cadavers. METHODS: Standard lateral shoulder arthroscopy was performed with or without the use of the Leipzig distractor (n = 15 each). Joint space width, procedure time, and visibility and palpability of the intra-articular structures were assessed during the arthroscopy. After the arthroscopic evaluation, each shoulder joint was disarticulated to assess the area and number of iatrogenic articular cartilage injury (IACI) lesions. Sites around the distraction device were assessed for the presence of iatrogenic injury. RESULTS: With shoulder distraction, median joint space width was 4 mm larger (P = .01), IACI area was 9.5 mm2 lower (P = .003), and there were two fewer total number of IACI lesions (P = .004) compared with nondistracted shoulders. The mean total surgery time was 93 seconds shorter (P = .01) in distracted shoulders. Although distraction was associated with increased visibility of the supraglenoid tubercle (P = .015), no significant differences were found for other intra-articular structures for their visibility and palpability. Unexpected lesions at the sites around the distraction device were not encountered. CONCLUSION: Use of the distraction device decreased the area and incidence of IACI lesions and shortened the arthroscopy time. However, no improvement was found in the visibility or palpability of the intra-articular structures. CLINICAL SIGNIFICANCE: Although additional clinical studies are required to evaluate the effect of the distraction device on pathologic articular conditions and intraoperative manipulation, the use of a shoulder distraction device might improve the outcomes of shoulder arthroscopy by decreasing IACI and arthroscopy time.

Polo-like kinase 4 and Stromal antigen 3 are not associated with recurrent pregnancy loss caused by embryonic aneuploidy.

No genetic association with recurrent pregnancy loss (RPL) caused by embryonic aneuploidy has been found. Recent studies have indicated that the common genetic variant rs2305957, surrounding the PLK4 gene, contributes to mitotic-origin aneuploidy risk during human early embryo development. The decrease in meiosis-specific cohesin causes predivision of sister chromatids in the centromere and chromosome segregation errors. STAG3 is a component of cohesin and is a meiosis-specific gene. Our case-control study included 184 patients with RPL whose previous products of conception (POC) exhibited aneuploidy and 190 fertile control women without a history of miscarriage. We performed a genetic association study to examine the genotype distribution at PLK4 (rs2305957) and STAG3 in patients with RPL caused by aneuploidy compared with controls. Regarding STAG3, SNPs with a minor allele frequency (MAF) threshold > 0.05 that were predicted to be binding sites of transcription factors and that showed significant associations in expression quantitative trait locus (e-QTL) analysis were selected. No significant differences in the MAF or distribution in any model of PLK4 (rs2305957) and 5 selected tag SNPs in STAG3 were found between the patients and controls. A further genome-wide association study is needed since a combination of genetic risk alleles might be useful in predicting future age-dependent RPL caused by aneuploidy.

Extrahepatic biliary duct obstruction secondary to duodenal foreign bodies in 2 dogs.

Two dogs, a 5-year-old neutered male boxer dog and a 2-year-old neutered male Australian cattle dog, were evaluated for vomiting and inappetence. Both dogs were icteric on physical examination, and had hyperbilirubinemia, elevated liver enzymes, and inflammatory changes on diagnostic blood analysis. A proximal duodenal obstruction causing extrahepatic biliary duct obstruction (EHBDO) was suspected in both cases after diagnostic imaging was performed. Exploratory laparotomy confirmed duodenal foreign bodies in both cases. This case report defines duodenal foreign body as a differential diagnosis for EHBDO in dogs.

Obstruction extra-hépatique de canaux biliaires secondaire à des corps étrangers duodénaux chez deux chiens. Deux chiens, un Boxer mâle castré âgé de 5 ans et un berger australien mâle castré âgé de 2 ans, furent évalués pour des vomissements et de l'inappétence. Les deux chiens étaient ictériques lors de l'examen physique, et l'analyse sanguine a montré la présence d'hyperbilirubinémie, de niveaux d'enzymes hépatiques élevés et de changements inflammatoires. Une obstruction duodénale proximale causant une obstruction extra-hépatique des canaux biliaires (EHBDO) était suspectée dans les deux cas à la suite des examens d'imagerie effectués. Une laparotomie exploratrice a confirmé la présence de corps étrangers duodénaux dans les deux cas. Ce rapport de cas identifie les corps étrangers duodénaux comme diagnostic différentiel lors d'EHBDO chez les chiens.(Traduit par Dr Serge Messier).

Blocking LC3 lipidation and ATG12 conjugation reactions by ATG7 mutant protein containing C572S.

Autophagy, a system for the bulk degradation of intracellular components, is essential for homeostasis and the healthy physiology and development of cells and tissues. Its deregulation is associated with human disease. Thus, methods to modulate autophagic activity are critical for analysis of its role in mammalian cells and tissues. Here we report a method to inhibit autophagy using a mutant variant of the protein ATG7, a ubiquitin E1-like enzyme essential for autophagosome formation. During autophagy, ATG7 activates the conjugation of LC3 (ATG8) with phosphatidylethanolamine (PE) and ATG12 with ATG5. Human ATG7 interactions with LC3 or ATG12 require a thioester bond involving the ATG7 cysteine residue at position 572. We generated TetOff cells expressing mutant ATG7 protein carrying a serine substitution of this critical cysteine residue (ATG7C572S). Because ATG7C572S forms stable intermediate complexes with LC3 or ATG12, its expression resulted in a strong blockage of the ATG-conjugation system and suppression of autophagosome formation. Consequently, ATG7C572S mutant protein can be used as an inhibitor of autophagy.

Effect of Bipolar Radiofrequency Energy on Canine Stifle Joint Fluid Temperature.

OBJECTIVE: The aim of this study was to evaluate the effect of bipolar radiofrequency (RF) energy on canine stifle joint fluid temperature. MATERIALS AND METHODS: A standard stifle arthroscopy was performed on 15 canine large breed cadaveric stifle joints. A bipolar RF (VAPR III, 2.3-mm side effect electrode; Depuy Mitek, Raynham, Massachusetts, United States) unit was activated in the joint (1) with or without direct tissue contact, (2) with or without additional 18-gauge needle outflow and (3) for 15 and 30 seconds. The joint fluid temperature was monitored with two fibre optic intra-articular sensors. RESULTS: The stifle joint fluid temperature was significantly higher when there was no contact between the tissue and RF probe (mean: 58.6°C with 95% confidence interval [CI]: 53.3-64.0°C) compared with when tissue was contacted (mean: 29.0°C with 95% CI: 26.3-31.6°C). An 18-gauge egress needle had minimal effect on reducing joint fluid temperature. The temperature was higher during the 30-second application of RF energy than the 15-second group. CLINICAL SIGNIFICANCE: Bipolar RF energy without firm tissue contact rapidly and significantly increased joint fluid temperature beyond the level reported to damage chondrocytes (above 45°C). Caution is required in the use of bipolar RF energy in the canine stifle joint.

Identification, expression analysis, and functional characterization of peptide YY in chickens (Gallus gallus domesticus).

Peptide YY (PYY) functions as a postprandial satiety signal in mammals. However, the genomic information and physiological roles of chicken PYY have not yet been clarified, although PYY peptide was isolated from chicken intestines in 1992. In this study, we identified a full-length complementary DNA (cDNA) sequence encoding the chicken PYY precursor. The deduced amino acid sequence of chicken PYY was completely consistent with the previously identified peptide sequence. PYY mRNA was abundantly expressed in the small intestine compared with the large intestine. PYY mRNA levels in the jejunum were significantly higher during ad libitum feeding compared with fasting, suggesting that intestinal PYY expression is altered in response to nutritional status in chicks. Intravenous administration of PYY significantly suppressed food intake in chicks. Furthermore, neuropeptide Y receptor Y2, a possible target of PYY, was expressed in various brain regions including the appetite-regulating centers in chicks. This is the first evidence that the intestinal hormone PYY may function as an anorexigenic hormone in chicks.

Reducing DNA methylation suppresses colon carcinogenesis by inducing tumor cell differentiation.

The forced reduction of global DNA methylation suppresses tumor development in several cancer models in vivo. Nevertheless, the mechanisms underlying these suppressive effects remain unclear. In this report, we describe our findings showing that a genome-wide reduction in the DNA methylation levels induces cellular differentiation in association with decreased cell proliferation in Apc (Min/+) mouse colon tumor cells in vivo. Colon tumor-specific DNA methylation at Cdx1 is reduced in the DNA-hypomethylated tumors accompanied by Cdx1 derepression and an increased expression of intestinal differentiation-related genes. Furthermore, a histological analysis revealed that Cdx1 derepression in the DNA-hypomethylated tumors is correlated with the differentiation of colon tumor cells. Similarly, the treatment of human colon cancer cell lines with a hypomethylating agent induces differentiation-related genes, including CDX1. We herein propose that DNA demethylation exerts a tumor suppressive effect in the colon by inducing tumor cell differentiation.

Mitochondrial function provides instructive signals for activation-induced B-cell fates.

During immune reactions, functionally distinct B-cell subsets are generated by stochastic processes, including class-switch recombination (CSR) and plasma cell differentiation (PCD). In this study, we show a strong association between individual B-cell fates and mitochondrial functions. CSR occurs specifically in activated B cells with increased mitochondrial mass and membrane potential, which augment mitochondrial reactive oxygen species (mROS), whereas PCD occurs in cells with decreased mitochondrial mass and potential. These events are consequences of initial slight changes in mROS in mitochondria(high) B-cell populations. In CSR-committed cells, mROS attenuates haeme synthesis by inhibiting ferrous ion addition to protoporphyrin IX, thereby maintaining Bach2 function. Reduced mROS then promotes PCD by increasing haeme synthesis. In PCD-committed cells, Blimp1 reduces mitochondrial mass, thereby reducing mROS levels. Identifying mROS as a haeme synthesis regulator increases the understanding of mechanisms regulating haeme homeostasis and cell fate determination after B-cell activation.

Intracerebroventricular administration of chicken glucagon-like peptide-2 potently suppresses food intake in chicks.

Glucagon-related peptides, such as glucagon-like peptide (GLP)-1, GLP-2 and oxyntomodulin (OXM), are processed from an identical precursor proglucagon. In mammals, all of these peptides are suggested to be involved in the central regulation of food intake. We previously showed that intracerebroventricular administration of chicken OXM and GLP-1 significantly suppressed food intake in chicks. Here, we show that central administration of chicken GLP-2 potently suppresses food intake in chicks. Male 8-day-old chicks (Gallus gallus domesticus) were used in all experiments. Intracerebroventricular administration of chicken GLP-2 significantly suppressed food intake in chicks. Plasma glucose concentration was significantly decreased by chicken GLP-2, whereas plasma nonesterified fatty acid concentration was significantly increased. Intracerebroventricular administration of chicken GLP-2 did not affect plasma corticosterone concentration. In addition, the anorexigenic effect of GLP-2 was not reversed by the corticotropin-releasing factor (CRF) receptor antagonist α-helical CRF, suggesting that CRF is not a downstream mediator of the anorexigenic pathway of GLP-2 in chicks. Intracerebroventricular administration of an equimolar amount of GLP-1 and GLP-2, but not OXM, significantly suppressed food intake in both broiler and layer chicks. All our findings suggest that GLP-2 functions as a potent anorexigenic peptide in the brain, as well as GLP-1, in chicks.

Correlation analysis of hypothalamic mRNA levels of appetite regulatory neuropeptides and several metabolic parameters in 28-day-old layer chickens.

Various lines of evidence suggest that appetite-related neuropeptides in the hypothalamus are regulated by adiposity signals such as leptin and insulin in mammals. In the present study, we examined age-dependent changes in the weight of abdominal fat and hypothalamic mRNA levels of neuropeptide Y (NPY, an orexigenic neuropeptide) and proopiomelanocortin (POMC, a precursor of anorexigenic neuropeptides) in growing chickens at 7, 14, 21 and 28 days of age. Hypothalamic NPY mRNA levels were significantly (P < 0.05) decreased after 14 days of age, whereas hypothalamic POMC mRNA levels were significantly (P < 0.05) increased at 28 days of age. The percentage of abdominal fat was significantly increased after 14 days of age in chickens. We next examined the correlation of hypothalamic NPY and POMC mRNA levels and several parameters at 28 days of age. There were no significant correlations between hypothalamic mRNA levels of NPY or POMC and the percentage of abdominal fat. These findings suggest that the gene expressions of NPY and POMC do not depend on adiposity in chickens, at least in 28-day-old layer chickens.

Intracerebroventricular administration of chicken oxyntomodulin suppresses food intake and increases plasma glucose and corticosterone concentrations in chicks.

Central administration of proglucagon-derived peptides, glucagon, glucagon-like peptide-1 (GLP-1), and oxyntomodulin (OXM), suppresses food intake in both mammals and birds. Recent findings suggest that GLP-1 receptor is involved in the anorexigenic action of OXM in both species. However, mammalian (bovine) OXM was used in chicken studies, even though the amino acid sequence and peptide length of chicken OXM differ from those of bovine OXM. In the present study, we examined the effect of chicken OXM on food intake and plasma components in chicks to investigate the mechanisms underlying the OXM effect. Male 8-day-old chicks (Gallus gallus domesticus) were used in all experiments. Intracerebroventricular administration of chicken OXM significantly suppressed food intake in chicks. Plasma concentrations of glucose and corticosterone were significantly increased by chicken OXM. These phenomena were also observed after bovine OXM injection in chicks. In contrast, central administration of chicken GLP-1 significantly decreased plasma glucose concentration and did not affect plasma corticosterone concentration. We previously showed that central administration of chicken glucagon significantly increased plasma concentrations of glucose and corticosterone in chicks. All our findings suggest that the mechanism underlying the anorexigenic action of OXM is similar to that of glucagon in chicks.

Intracerebroventricular administration of novel glucagon-like peptide suppresses food intake in chicks.

Glucagon-related peptides such as glucagon, glucagon-like peptide-1, and oxyntomodulin suppress food intake in mammals and birds. Recently, novel glucagon-like peptide (GCGL) was identified from chicken brain, and a comparatively high mRNA expression level of GCGL was detected in the hypothalamus. A number of studies suggest that the hypothalamus plays a critical role in the regulation of food intake in mammals and birds. In the present study, we investigated whether GCGL is involved in the central regulation of food intake in chicks. Male 8-day-old chicks (Gallus gallus) were used in all experiments. Intracerebroventricular administration of GCGL in chicks significantly suppressed food intake. Plasma glucose level was significantly decreased by GCGL, whereas plasma corticosterone level was not affected. Central administration of a corticotrophin-releasing factor (CRF) receptor antagonist, α-helical CRF, attenuated GCGL-suppressed food intake. It seems likely that CRF receptor is involved in the GCGL-induced anorexigenic pathway. All our findings suggest that GCGL functions as an anorexigenic peptide in the central nervous system of chicks.

Characterization of regulatory T cells in decidua of miscarriage cases with abnormal or normal fetal chromosomal content.

Decreased regulatory T (Treg) cells have been reported in cases of recurrent pregnancy loss. To understand the role of Treg cells in human pregnancy, we have studied the frequency, localization and characterization of Treg cells in the decidua. The frequency of Foxp3(+) cells among CD3(+)CD8(-) cells at the decidua basalis in cases of miscarriage with a normal embryo karyotype (n=10) was significantly lower than in normally progressing pregnancies (n=10). However, those frequencies in miscarriage with an abnormal embryo karyotype were similar to normally progressing pregnancies. Next, we used flow cytometry to study Treg cell expression of the proliferation marker Ki67 and functional Treg marker CCR5. The frequency of Foxp3(+)CD4(+) T cells in miscarriage with a normal embryo (n=10) was significantly lower than those in normally progressing pregnancies (n=15) and in miscarriage with an abnormal embryo (n=14). In miscarriage with a normal embryo, the population of Ki67(-)Foxp3(+)CD4(+) T cells was significantly smaller than in normal pregnancy. However, the frequencies of Ki67(+)Foxp3(+)CD4(+) cells and CCR5(+)Foxp3(+)CD4(+) cells were not different between the three groups. These data suggest that increased Ki67(-) Treg cells in the decidua basalis may play an important role in the induction of immune tolerance, and that immune-medicated pregnancy loss may be caused by decreased Ki67(-) Treg cells in the implantation site.