The fluid factor OVGP1 provides a significant oviductal microenvironment for the reproductive process in golden hamster†.

The mammalian oviductal lumen is a specialized chamber that provides an environment that strictly regulates fertilization and early embryogenesis, but the regulatory mechanisms to gametes and zygotes are unclear. We evaluated the oviductal regulation of early embryonic development using Ovgp1 (encoding an oviductal humoral factor, OVGP1)-knockout golden hamsters. The experimental results revealed the following: (1) female Ovgp1-knockout hamsters failed to produce litters; (2) in the oviducts of Ovgp1-knockout animals, fertilized eggs were sometimes identified, but their morphology showed abnormal features; (3) the number of implantations in the Ovgp1-knockout females was low; (4) even if implantations occurred, the embryos developed abnormally and eventually died; and (5) Ovgp1-knockout female ovaries transferred to wild-type females resulted in the production of Ovgp1-knockout egg-derived OVGP1-null litters, but the reverse experiment did not. These results suggest that OVGP1-mediated physiological events are crucial for reproductive process in vivo, from fertilization to early embryonic development. This animal model shows that the fate of the zygote is determined not only genetically, but also by the surrounding oviductal microenvironment.

Targeting of neutrophil activation in the early phase of the disease for prevention of Coronavirus disease-19 severity.

The prevention of the disease severity seems critical for reducing the mortality of Coronavirus (CoV) disease-19. The neutrophils play a key role in the induction of severity. It is proposed here that inhibition of neutrophil activation and/or cascade reactions of complement, leading to this cell activation at the early phase of the disease, is a potential tool to inhibit aggravation of the disease. The need for appropriate timing in intervention is emphasized as follows. (1) Intervention at the very early stage of severe acute respiratory syndrome-CoV-2 infection may harm the defensive host response to the infection because of the critical function of neutrophils in this response, and (2) intervention at too late a stage will not stop the infiltration of fully activated neutrophils that produce large amounts of toxic substances.

White matter alterations in the dorsal attention network contribute to a high risk of unsafe driving in healthy older people.

Aim: Healthy older drivers may be at high risk of fatal traffic accidents. Our recent study showed that volumetric alterations in gray matter in the brain regions within the dorsal attention network (DAN) were strongly related to the risk of unsafe driving in healthy older people. However, the relationship between white matter (WM) structural connectivity and driving ability in healthy older people is still unclear. Methods: We used diffusion tensor imaging to examine the association between microstructural alterations in the DAN and the risk of unsafe driving among healthy older people. We enrolled 32 healthy older individuals aged over 65 years and screened unsafe drivers using an on-road driving test. We then determined the pattern of WM aberrations in unsafe drivers using tract-based spatial statistics. Results: The analysis demonstrated that unsafe drivers had significantly higher axial diffusivity values in nine WM clusters compared with safe drivers. These results were primarily observed bilaterally in the dorsal superior longitudinal fasciculus, which is involved in the DAN. Furthermore, correlation analyses showed that higher axial diffusivity values in the superior longitudinal fasciculus were associated with lower Trail Making Test A scores within unsafe drivers. This result suggests that functionally, WM microstructural alterations in the DAN are associated with attention problems, which may contribute to the risk of unsafe driving among healthy older people. Conclusion: Our findings may elucidate the neurobiological mechanisms underlying the increased risk of unsafe driving in healthy older people, potentially facilitating the development of new interventions to prevent fatal accidents.

An overview of sex and reproductive immunity from an evolutionary/anthropological perspective.

Mammalian pregnancy is a curious life phenomenon. Immunologically, the mechanism of pregnancy is difficult to explain because it involves the coexistence of an external foreign body (the embryo) and the host (the mother) for a period of time. How did mammals acquire the ability to become pregnant in parallel with altered immunity? Sex in the evolution of life and its impact on anthropology are major topics of discussion. In this paper, we outline (1) sex and evolution in mammals after the advent of our direct ancestors (apes) up to humans (i.e., the Cenozoic Quaternary), including anthropological aspects such as the development of the central nervous system; (2) the development of reproductive immunity during the Paleozoic era, when biodiversity developed explosively (and many sexually reproducing organisms have emerged); and (3) the characteristic reproductive strategies of mammals, including humans with the immunological aspects of viviparity. We present an overview of mammalian reproductive immunity, which is a heretical aspect of immunology.

Regional Gray Matter Volume Identifies High Risk of Unsafe Driving in Healthy Older People.

In developed countries, the number of traffic accidents caused by older drivers is increasing. Approximately half of the older drivers who cause fatal accidents are cognitively normal. Thus, it is important to identify older drivers who are cognitively normal but at high risk of causing fatal traffic accidents. However, no standardized method for assessing the driving ability of older drivers has been established. We aimed to establish an objective assessment of driving ability and to clarify the neural basis of unsafe driving in healthy older people. We enrolled 32 healthy older individuals aged over 65 years and classified unsafe drivers using an on-road driving test. We then utilized a machine learning approach to distinguish unsafe drivers from safe drivers based on clinical features and gray matter volume data. Twenty-one participants were classified as safe drivers and 11 participants as unsafe drivers. A linear support vector machine classifier successfully distinguished unsafe drivers from safe drivers with 87.5% accuracy (sensitivity of 63.6% and specificity of 100%). Five parameters (age and gray matter volume in four cortical regions, including the left superior part of the precentral sulcus, the left sulcus intermedius primus [of Jensen], the right orbital part of the inferior frontal gyrus, and the right superior frontal sulcus), were consistently selected as features for the final classification model. Our findings indicate that the cortical regions implicated in voluntary orienting of attention, decision making, and working memory may constitute the essential neural basis of driving behavior.

Role of the Glycosylphosphatidylinositol-Anchored Protein TEX101 and Its Related Molecules in Spermatogenesis.

Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) on the plasma membrane are involved in several cellular processes, including sperm functions. Thus far, several GPI-APs have been identified in the testicular germ cells, and there is increasing evidence of their biological significance during fertilization. Among GPI-APs identified in the testis, this review focuses on TEX101, a germ cell-specific GPI-AP that belongs to the lymphocyte antigen 6/urokinase-type plasminogen activator receptor superfamily. This molecule was originally identified as a glycoprotein that contained the antigen epitope for a specific monoclonal antibody; it was produced by immunizing female mice with an allogenic testicular homogenate. This review mainly describes the current understanding of the biochemical, morphological, and physiological characteristics of TEX101. Furthermore, future avenues for the investigation of testicular GPI-Aps, including their potential role as regulators of ion channels, are discussed.

Machine-learning approach to predict on-road driving ability in healthy older people.

AIM: In Japan, fatal traffic accidents due to older drivers are on the rise. Considering that approximately half the older drivers who have caused fatal accidents are cognitively normal healthy people, it has been required to detect older drivers who are cognitively normal but at high risk of having fatal traffic accidents. However, a standardized method for assessing the driving ability of older drivers has not yet been established. We thus aimed to identify a new sensing method for the evaluation of the on-road driving ability of healthy older people on the basis of vehicle behaviors. METHODS: We enrolled 33 healthy older individuals aged over 65 years and utilized a machine-learning approach to dissociate unsafe drivers from safe drivers based on cognitive assessments and a functional visual acuity test. RESULTS: The linear support vector machine classifier successfully dissociated unsafe drivers from safe drivers with accuracy of 84.8% (sensitivity of 66.7% and specificity of 95.2%). Five clinical parameters, namely age, the first trial of the Rey Auditory Verbal Learning Test immediate recall, the delayed recall of the Rey-Osterrieth Complex Figure Test, the result of the free-drawn Clock Drawing Test, and maximal visual acuity, were consistently selected as essential features for the best classification model. CONCLUSION: Our findings improve our understanding of clinical risk factors leading to unsafe driving and may provide insight into a new intervention that prevents fatal traffic accidents caused by healthy older people.

NUP62: the target of an anti-sperm auto-monoclonal antibody during testicular development.

Ts4, an autosperm-monoclonal antibody (mAb), reacts with a specific oligosaccharide (OS) of glycoproteins containing bisecting N-acetylglucosamine residues. Ts4 reactivity was observed against epididymal spermatozoa, testicular germ cells, and the early embryo, but not against major organs in adult mice. In mature testis, Ts4 exhibits immunoreactivity with a germ cell-specific glycoprotein, TEX101, whereas the mAb immunoreacts with alpha-N-acetylglucosaminidase in the acrosomal region of cauda epididymal spermatozoa. Thus, Ts4 seems to react against different molecules throughout spermiogenesis via binding to its OS epitope. Since the Ts4-epitope OS is observed only in reproduction-related regions, the Ts4-reactive OS may play a role in the reproductive process. The aim of this study is to investigate the characteristics of the Ts4-reactive molecule(s) during testicular development. Ts4 reactivity was observed in testes from the prenatal period; however, its distribution changed according to the stage of maturation and was identical to that of the adult testes after 29-day-postpartum (dpp). Ts4 immunoreactivity was detected against a protein with 63 kDa in testis from 1 to 29 dpp. In contrast, Ts4 showed reactivity against some other glycoproteins after 29 dpp, including TEX101 at the 5-week-old stage and onward. To identify the Ts4-reactive 63 kDa molecule, we identified NUP62 as the target of Ts4 in 22 dpp testis using liquid chromatography-tandem mass spectrometry analysis. Because NUP62 has been known to play active roles in a variety of cellular processes including mitosis and cell migration, the bisecting GlcNAc recognized by Ts4 on NUP62 may play a role in regulating the early development of germ cells in male gonadal organs.

Quantitative measurement of a candidate serum biomarker peptide derived from α2-HS-glycoprotein, and a preliminary trial of multidimensional peptide analysis in females with pregnancy-induced hypertension.

Purpose We previously attempted to develop quantitative enzyme-linked immunosorbent assay (ELISA) systems for the PDA039/044/071 peptides, potential serum disease biomarkers (DBMs) of pregnancy-induced hypertension (PIH), primarily identified by a peptidomic approach (BLOTCHIP®-mass spectrometry (MS)). However, our methodology did not extend to PDA071 (cysteinyl α2-HS-glycoprotein341-367), due to difficulty to produce a specific antibody against the peptide. The aim of the present study was to establish an alternative PDA071 quantitation system using liquid chromatography-multiple reaction monitoring (LC-MRM)/MS, to explore the potential utility of PDA071 as a DBM for PIH. Methods We tested heat/acid denaturation methods in efforts to purify serum PDA071 and developed an LC-MRM/MS method allowing for specific quantitation thereof. We measured serum PDA071 concentrations, and these results were validated including by three-dimensional (3D) plotting against PDA039 (kininogen-1439-456)/044 (kininogen-1438-456) concentrations, followed by discriminant analysis. Results PDA071 was successfully extracted from serum using a heat denaturation method. Optimum conditions for quantitation via LC-MRM/MS were developed; the assayed serum PDA071 correlated well with the BLOTCHIP® assay values. Although the PDA071 alone did not significantly differ between patients and controls, 3D plotting of PDA039/044/071 peptide concentrations and construction of a Jackknife classification matrix were satisfactory in terms of PIH diagnostic precision. Conclusions Combination analysis using both PDA071 and PDA039/044 concentrations allowed PIH diagnostic accuracy to be attained, and our method will be valuable in future pathophysiological studies of hypertensive disorders of pregnancy.

Involvement of cross-reactive carbohydrate determinants-specific IgE in pollen allergy testing.

BACKGROUND: Specific IgE antibodies against the low-molecular-weight carbohydrate antigen that does not bridge IgE molecules on mast cells are not associated with clinical symptoms. Cross reactivity can be determined in allergen-specific IgE detection assays when the carbohydrate structures between pollen allergens and plant derived food allergens are similar; in such cases, false positive results for grain or legume allergens can be reported for pollen allergic patients who are not sensitized to those allergens. This phenomenon arises owing to the presence of cross-reactive carbohydrate determinants (CCDs). OBJECTIVE: This study aimed to assess the impact of CCD interference on the results for pollen allergen-specific IgE antibodies in the general adult population and to perform CCD inhibition tests evaluating the involvement of CCD on samples positive to pollen allergens. METHODS: Serum samples from 322 subjects were tested for IgE antibodies to pollens and CCD. The research subjects were given questionnaires about pollen allergic symptoms to help assess the presence of allergies. Allergen IgE antibodies for Japanese cedar, Japanese cypress, orchard grass, ragweed, MUXF, bromelain, horseradish peroxidase (HRP), and ascorbate oxidase (ASOD) were analyzed. RESULTS: It was observed that among individuals who tested positive to any of the pollen allergens, the positive ratio of CCD-specific IgE antibody was the highest for HRP (13.5%-50.0%). The results from the inhibition tests revealed that CCD was marginally present. Although IgE antibodies for cedar pollen did not react with CCD, IgE antibodies for Japanese cypress, orchard grass, and ragweed might be detected by the presence of CCD. CONCLUSION: The results of the inhibition tests revealed the obvious presence of CCD suggesting its involvement. Considering these findings, careful evaluation of patient IgE results should be performed for Japanese cypress, orchard grass, and ragweed.

Identification of an anti-sperm auto-monoclonal antibody (Ts4)-recognized molecule in the mouse sperm acrosomal region and its inhibitory effect on fertilization in vitro.

We previously established an anti-mouse sperm auto-monoclonal antibody, Ts4, which shows immunoreactivity against several kinds of glycoproteins in the acrosomal region of epididymal spermatozoa, testicular germ cells, and early embryo, via binding to an epitope containing a common N-linked oligosaccharide (OS) chain on the molecules. In mice, we have already demonstrated that the OS chain in the epitope for Ts4 is a fucosylated agalacto-complex-type biantennary glycan carrying bisecting N-acetylglucosamine. In the testis, one of the specific OS chain-conjugated molecules is TEX101, a germ cell-marker glycoprotein, which is expressed in spermatocytes, spermatids, and testicular spermatozoa, but not in epididymal spermatozoa. In this study, we identified a Ts4-reactive glycoprotein in mouse cauda epididymal sperm. An immunoprecipitation method together with liquid chromatography-tandem mass spectrometry showed that alpha-N-acetylglucosaminidase (Naglu; a degradation enzyme of heparan sulfate) is one of the glycoproteins recognized by Ts4 in the epididymal spermatozoa. Western blot and immunohistochemical analyses revealed that mouse Naglu exists in two forms (82 and 77kDa) and is expressed in the acrosomal region and the flagellum of cauda epididymal sperm. Of the two Naglu-forms expressed in sperm, Ts4 immunoreacted against only the 82-kDa form located on the acrosomal region. The Ts4 mAb and anti-Naglu pAb negatively affected mouse fertilization in vitro. In addition, Ts4 inhibited sperm acrosome reaction induced by heparan sulfate. The Ts4-recognized fucosylated agalactobiantennary complex-type glycan with bisecting N-acetylglucosamine and Naglu on cauda epididymal spermatozoa may play a role in the process of fertilization.

TEX101, a glycoprotein essential for sperm fertility, is required for stable expression of Ly6k on testicular germ cells.

TEX101, a germ cell-specific glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein, is associated with Ly6k during spermatogenesis in testis. Although both Tex101(-/-) and Ly6k(-/-) mice can produce morphologically intact spermatozoa, both knockout mice show an infertile phenotype due to a disorder of spermatozoa to migrate into the oviduct. Since Ly6k specifically interacts with TEX101, complex formation of TEX101/Ly6k appears to be potentially important for functional sperm production. This study evaluated the fate of Ly6k in the presence or absence of TEX101 to explore the molecular interaction of both GPI-anchored proteins in seminiferous tubules. The present study showed that: 1) Although Ly6k mRNA was detected, the protein was present at very low levels in mature testes of Tex101(-/-) mice, 2) Ly6k mRNA level was within the normal range in Tex101(-/-) mice, 3) Ly6k mRNA was translated into a polypeptide in the testes of Tex101(+/+) and Tex101(-/-) mice, and 4) TEX101, as well as Ly6k, are co-factors that affect to molecular expression. These results indicate that both TEX101 and Ly6k contribute to the post-translational counterpart protein expression at the cell membrane. This mechanism may be important in maintaining the production of fertile spermatozoa during spermatogenesis.

Chemical Characterization of N-Linked Oligosaccharide As the Antigen Epitope Recognized by an Anti-Sperm Auto-Monoclonal Antibody, Ts4.

Ts4, an anti-sperm auto-monoclonal antibody, possesses immunoreactivity to the acrosomal region of mouse epididymal spermatozoa. In addition, the mAb shows specific immunoreactivity to reproduction-related regions such as testicular germ cells and early embryo. Our qualitative study previously showed that the antigen epitope for Ts4 contained a N-linked common oligosaccharide (OS) chain on testicular glycoproteins as determined by Western blotting for testicular glycoproteins after treatment with several glycohydrolases. Since the distribution of the Ts4-epitope is unique, the OS chain in Ts4-epitope may have role(s) in the reproductive process. The aim of this study was to clarify the molecular structure of the Ts4-epitope, particularly its OS moiety. Using Ts4 immunoprecipitation combined with liquid chromatography and multiple-stage mass spectrometry, the candidate carbohydrate structure in the Ts4-epitope is proposed to be N-linked fucosylated agalacto-biantennary with bisecting N-acetylglucosamine (GlcNAc) or with N-acetylgalactosamine-GlcNAc motif. Further binding analyses using various lectins against the mouse testicular Ts4-immunoprecipitants revealed that Phaseolus vulgaris erythroagglutinin and Pisum sativum agglutinin showed positive staining of the bands corresponding to Ts4 reactive proteins. Moreover, the immunoreactivity of Ts4 against the testicular extract was completely abrogated after digestion with ß-N-acetylglucosaminidase. These results show that the Ts4-epitope contains agalacto-biantennary N-glycan with bisecting GlcNAc carrying fucose residues.

Maternal peripheral blood natural killer cells incorporate placenta-associated microRNAs during pregnancy.

Although recent studies have demonstrated that microRNAs (miRNAs or miRs) regulate fundamental natural killer (NK) cellular processes, including cytotoxicity and cytokine production, little is known about the miRNA-gene regulatory relationships in maternal peripheral blood NK (pNK) cells during pregnancy. In the present study, to determine the roles of miRNAs within gene regulatory networks of maternal pNK cells, we performed comprehensive miRNA and gene expression profiling of maternal pNK cells using a combination of reverse transcription quantitative PCR (RT-qPCR)-based miRNA array and DNA microarray analyses and analyzed the differential expression levels between first- and third-trimester pNK cells. Furthermore, we constructed regulatory networks for miRNA-mediated gene expression in pNK cells during pregnancy by Ingenuity Pathway Analysis (IPA). PCR-based array analysis revealed that the placenta-derived miRNAs [chromosome 19 miRNA cluster (C19MC) miRNAs] were detected in pNK cells during pregnancy. Twenty-five miRNAs, including six C19MC miRNAs, were significantly upregulated in the third- compared to first-trimester pNK cells. The rapid clearance of C19MC miRNAs also occurred in the pNK cells following delivery. Nine miRNAs, including eight C19MC miRNAs, were significantly downregulated in the post-delivery pNK cells compared to those of the third-trimester. DNA microarray analysis identified 69 NK cell function-related genes that were differentially expressed between the first- and third-trimester pNK cells. On pathway and network analysis, the observed gene expression changes of pNK cells likely contribute to the increase in the cytotoxicity, as well as the cell cycle progression of third- compared to first-trimester pNK cells. Thirteen of the 69 NK cell function-related genes were significantly downregulated between the first- and third-trimester pNK cells. Nine of the 13 downregulated NK-function-associated genes were in silico target candidates of 12 upregulated miRNAs, including C19MC miRNA miR-512-3p. The results of this study suggest that the transfer of placental C19MC miRNAs into maternal pNK cells occurs during pregnancy. The present study provides new insight into maternal NK cell functions.

Human exosomal placenta-associated miR-517a-3p modulates the expression of PRKG1 mRNA in Jurkat cells.

During pregnancy, human placenta-associated microRNAs (miRNAs) derived from the miRNA cluster in human chromosome 19 are expressed in villous trophoblasts and secreted into maternal circulation via exosomes; however, little is known about whether circulating placenta-associated miRNAs are transferred into maternal immune cells via exosomes, and modulate expression of target genes in the recipient cells. We employed an in vitro model of trophoblast-immune cell communication using BeWo cells (a human trophoblast cell line) and Jurkat cells (a human leukemic T-cell line) and investigated whether BeWo exosomal placenta-associated miRNAs can suppress expression of target genes in the recipient Jurkat cells. Using this system, we identified PRKG1 as a target gene of placenta-associated miRNA miR-517a-3p. Moreover, we demonstrated that BeWo exosomal miR-517a-3p was internalized into Jurkat cells and subsequently suppressed the expression of PRKG1 in recipient Jurkat cells. Furthermore, using peripheral blood natural killer (NK) cells in vivo, we confirmed that circulating miR-517a-3p was delivered into maternal NK cells as it was into Jurkat cells in vitro. Placenta-associated miR-517a-3p was incorporated into maternal NK cells in the third trimester, and it was rapidly cleared after delivery. Expression levels of miR-517a-3p and its target mRNA PRKG1 were inversely correlated in NK cells before and after delivery. These in vitro and in vivo results suggest that exosome-mediated transfer of placenta-associated miRNAs and subsequent modulation of their target genes occur in maternal NK cells. The present study provides novel insight into our understanding of placenta-maternal communication.

Postnatal epigenetic modification of glucocorticoid receptor gene in preterm infants: a prospective cohort study.

OBJECTIVE: To examine the environmental effects on cytosine methylation of preterm infant's DNA, because early life experiences are considered to influence the physiological and mental health of an individual through epigenetic modification of DNA. DESIGN: A prospective cohort study, comparison of epigenetic differences in the glucocorticoid receptor (GR) gene between healthy term and preterm infants. SETTING: Neonatal Intensive Care Unit in a Japanese University Hospital. PARTICIPANTS: A cohort of 40 (20 term and 20 preterm) infants was recruited on the day of birth, and peripheral blood was obtained from each infant at birth and on postnatal day 4. MAIN OUTCOME MEASURES: The methylation rates in the 1-F promoter region of the GR gene using the Mquant method. RESULTS: The methylation rate increased significantly between postnatal days 0 and 4 in preterm infants but remained stable in term infants. Thus, the methylation rate was significantly higher in preterm than in term infants at postnatal day 4. Several perinatal parameters were significantly correlated with this change in the methylation rate. Logistic regression analysis revealed that methylation rates at postnatal day 4 predicted the occurrence of later complications that required glucocorticoid administration during the neonatal period. No gene polymorphism was detected within the GR promoter region analysed. CONCLUSIONS: Although further large-scale studies are needed to detect the environmental factors that explain the difference in epigenetic modification among infants after birth, our data show that the postnatal environment influences epigenetic programming of GR expression through methylation of the GR gene promoter in premature infants, which may result in relative glucocorticoid insufficiency during the postnatal period.

Placenta-specific miRNA (miR-512-3p) targets PPP3R1 encoding the calcineurin B regulatory subunit in BeWo cells.

AIM: The microRNAs (miRNAs) derived from the chromosome 19 miRNA cluster (C19MC) are exclusively expressed in the human placenta, but the origin and functions of C19MC miRNAs are not fully understood. The purpose of this study was to elucidate which cells express C19MC miRNAs in chorionic villi and identify their miRNA targets. METHODS: A combination of laser microdissection (LMD) and real-time polymerase chain reaction (PCR) to examine the localization of five C19MC miRNAs (i.e. miR-512-3p, miR-518b, miR-520a, miR-524 and miR-1323) in the human placenta was performed. Furthermore, to identify miR-512-3p-target genes, we analyzed gene expression profiles of the trophoblast cell line BeWo using a DNA microarray. Predicted target genes were validated by real-time PCR, western blotting, and 3'-untranslated region reporter assay. RESULTS: By LMD and subsequent PCR analysis, five C19MC miRNAs examined in this study were predominantly expressed in villous trophoblast cells; little expression, if any, was observed in villous stroma cells or fetal endothelial cells. Microarray data showed that 334 genes were downregulated in BeWo cells treated with Pre-miR-512-3p (mature miR-512-3p mimic). We found six candidate target genes of miR-512-3p using DNA microarray data and target prediction software. Furthermore, we revealed that protein phosphatase 3, regulatory subunit B, alpha (PPP3R1), one of the six genes, was a miR-512-3p target using an in vitro experimental validation system. CONCLUSION: These data suggest that miR-512-3p participates in human trophoblast function[s] by targeting PPP3R1, encoding a regulatory subunit of calcineurin.

The miR-221/222 cluster, miR-10b and miR-92a are highly upregulated in metastatic minimally invasive follicular thyroid carcinoma.

Minimally invasive follicular thyroid carcinoma (MI-FTC) is characterized by limited capsular and/or vascular invasion with good long-term outcomes. However, some cases of MI-FTC show a poor prognosis because of severe distant metastasis (i.e., metastatic MI-FTC). Nonetheless, no method has been established for predicting the prognosis of MI-FTC. This study was conducted to identify novel prognostic factors for metastatic MI-FTC by the use of microRNA (miRNA). Thirty-four patients with MI-FTC were categorized into two groups: the metastatic group, M(+) (n=12) and the non-metastatic group, M(-) (n=22). In the M(+) group, distant metastasis was recognized after the initial operation established the diagnosis of MI-FTC. In the M(-) group, no distant metastasis was recognized postoperatively for ≥ 10 years. Using laser microdissection followed by quantitative real-time PCR and PCR arrays, we performed a comprehensive expression profiling of 667 miRNAs in formalin-fixed, paraffin-embedded samples from the initial MI-FTC operation. Furthermore, we assessed the potential use of miRNAs as novel biomarkers for the metastatic potential of MI-FTC by logistic regression analysis. Comprehensive quantitative analysis of miRNA expression in MI-FTC samples revealed that the miR-221/222 cluster (i.e., miR-221, miR-222 and miR-222*), miR-10b and miR-92a were significantly upregulated in the M(+) group compared with the M(-) group. Interestingly, the expression levels of these miRNAs were also shown to be upregulated in widely invasive FTC (WI-FTC; n=13) that has distant metastasis and worse prognosis, indicating a close similarity in the miRNA expression between metastatic MI-FTC and WI-FTC. Logistic regression analysis revealed that miR-10b made a significant contribution to prognosis (OR 19.759, 95% CI 1.433-272.355, p=0.026). Our findings suggest that miR-10b is a potential prognostic factor for evaluating the metastatic potential of MI-FTC at an initial operation stage.

Overexpression of TEX101, a potential novel cancer marker, in head and neck squamous cell carcinoma.

TEX101, a member of the Ly-6/urokinase-type plasminogen activator receptor (LU)-family we previously identified, is a germ cell-marker glycoprotein. To date, it is reported that some members of the protein-family are overexpressed in a variety of cancer tissues. We previously reported Ly6k, a member of the LU-family, as an association molecule with TEX101 in murine male germ cells. LY6K (a human homologue of Ly6k) is overexpressed in head and neck squamous cell carcinomas (HNSCC). These facts led us to speculate that TEX101 may also exist in HNSCC, like LY6K. Using an anti-human TEX101 polyclonal antibody (pAb) established, we examined the expression of TEX101 protein in cancer tissues by immunohistochemical analysis. TEX101 was detected in the cancer cells of some tissue specimens from patients with HNSCC, whereas the normal squamous epithelium was immunonegative. The TEX101 protein was detected in cancer cells from 54 out of 64 (80.6%) patients with HNSCC. The rate of lymph nodes metastasis tends to be low in TEX101-positive patients, compared to patients with weakly positive and negative expression of TEX101. The present results imply that TEX101 is a novel cancer-related protein and may be useful as a marker for prognosis/diagnosis of HNSCC.

Molecular characterization and expression of dipeptidase 3, a testis-specific membrane-bound dipeptidase: complex formation with TEX101, a germ-cell-specific antigen in the mouse testis.

We previously established an anti-sperm head auto-monoclonal antibody designated Ts4. The immunoreactivity of this antibody was also observed in other reproduction-related cells, such as testicular germ cells and early embryos, suggesting that the Ts4-recognized molecules might play a role in the reproductive process. However, the molecular characteristics and functions of the antigens warrant further clarification. In this study, we primarily attempted identification of the mAb-recognized molecules within the mouse testis. An immunoprecipitation method, together with liquid chromatography-tandem mass spectrometry, revealed that the testicular immunoprecipitants with Ts4 contained dipeptidase 3 (DPEP3), a member of the membrane-bound dipeptidase family. A Western blot analysis using an anti-DPEP3 polyclonal antibody established in this study showed that this molecule was glycosylated and formed a disulfide-linked homodimer within the testis. Expression of DPEP3 protein was observed in the testicular germ cells, but not in the Sertoli or interstitial cells, or in any other major organs. Although Western blot analysis of testicular proteins separated by two-dimensional SDS-PAGE failed to demonstrate binding of Ts4 to DPEP3, we found that DPEP3 forms complexes with Ts4-immunoreactive molecules, such as TEX101, on the surfaces of spermatocytes, spermatids, and testicular spermatozoa. Based on data showing in the present study, further studies concerning DPEP3 on the testicular germ cells may help to clarify the molecular mechanisms of testicular germ-cell development.