Relationship between the timing of the first postpartum ovulation and antral follicle counts in Holstein cows.

BACKGROUND: The timing of the first postpartum ovulation is an important factor affecting the timing of estrous resumption in dairy cows. The first postpartum ovulation is delayed in cows producing large amounts of milk with an intensive negative energy balance. The antral follicle count (AFC) and serum anti-Müllerian hormone concentrations are known to be indicators of the ovarian reserve, which is the number and quality of follicles left in a pair of ovaries and known as an indicator of female fertility. Cows with higher AFC have been proven to show higher pregnancy rate and shorter calving to conception intervals; however, the relationship between the timing of the first postpartum ovulation and ovarian reserve remains unclear. Therefore, this study examined the relationships between postpartum follicular dynamics, the ovarian cycle, nutritional status, and ovarian reserve. METHODS: Transrectal ultrasonography was conducted from calving to 70-120 days in milk (DIM) in 26 cows to monitor AFC, follicular dynamics and the ovarian cycle. Body weight (BW) and milk yield were used as indicators of nutritional status. RESULTS: The first postpartum ovulation was significantly later in cows with low AFC (< 25) than in those with high AFC (≥25), while changes in BW from calving to the nadir and milk production were similar in both groups. The present results also suggested that cows with low AFC and a delayed first postpartum ovulation had a shorter first ovarian cycle after the first postpartum ovulation. The mean DIM of the first postpartum artificial insemination (AI) and days open (days from calving to AI with which pregnancy was achieved) were similar in high and low AFC groups. CONCLUSIONS: The first postpartum ovulation was significantly earlier in cows with high AFC than in those with low AFC. The assumed reason for this result was higher sensitivity to luteinizing hormone and larger androstenedione and estradiol production in follicles in high AFC cows. Therefore, cows with high AFC may be more fertile than those with low AFC while their milk production increase and BW decrease; it means they are in negative energy balance. (340/350 words).

[A middle to long-term clinical study on pars flaccida and pars tensa cholesteatoma using survival analysis].

The current clinical study was performed on 311 cases of pars flaccida and 89 cases of pars tensa cholesteatoma which were treated with canal wall reconstructed tympanoplasty between 1991 and 2012. The average follow-up time of these patients was 5.3 years. Since follow-up periods were different in each case and some censored patients were involved, we used survival analysis on this study to discuss the cumulative rates of disease-free successful cases and the rates of recurrent cholesteatoma throughout the postoperative course. The disease-free successful cases were defined as those cases in which patients were both out of re-operation with recurrent and residual cholesteatoma and out of revision operation with another problem, furthermore, maintained good hearing outcome. Based on the criteria set by the Japan Otological Society (2010), the cases that satisfied the following were evaluated as good hearing results; (a) a successful case in which preoperative bone conduction was used, and (b) a case in which the postoperative air-bone gap was within 20dB after tympanoplasty for chronic otitis media. The analysis results were shown for each of (a) and (b). 1. In pars flaccida cholesteatoma, the 5-year survival rate of successful case was (a) 76.1% and (b) 83.9%, the 10-year survival rate was (a) 58.9% and (b) 73.0%. In pars tensa cholesteatoma, the 5-year survival rate of successful cases was (a) 57.7% and (b) 63.5%, the 10-year rate was (a) 42.1% and (b) 56.9%. A significant difference was seen between pars flaccida and pars tensa cholesteatoma (p < 0.001). 2. In pars flaccida cholesteatoma, the 5-year recurrence rate was 7.6% and the 10-year rate was 15.3%, and the recurrence rate increased gradually throughout the follow-up period. On the other hand, in pars tensa cholesteatoma, the increase in the recurrence rate reached a peak 15.8% at 5.5 years after the surgery. A long-term follow-up is necessary when evaluating the clinical results after tympanoplasty.

Curcumin prevents replication of respiratory syncytial virus and the epithelial responses to it in human nasal epithelial cells.

The human nasal epithelium is the first line of defense during respiratory virus infection. Respiratory syncytial virus (RSV) is the major cause of bronchitis, asthma and severe lower respiratory tract disease in infants and young children. We previously reported in human nasal epithelial cells (HNECs), the replication and budding of RSV and the epithelial responses, including release of proinflammatory cytokines and enhancement of the tight junctions, are in part regulated via an NF-κB pathway. In this study, we investigated the effects of the NF-κB in HNECs infected with RSV. Curcumin prevented the replication and budding of RSV and the epithelial responses to it without cytotoxicity. Furthermore, the upregulation of the epithelial barrier function caused by infection with RSV was enhanced by curcumin. Curcumin also has wide pharmacokinetic effects as an inhibitor of NF-κB, eIF-2α dephosphorylation, proteasome and COX2. RSV-infected HNECs were treated with the eIF-2α dephosphorylation blocker salubrinal and the proteasome inhibitor MG132, and inhibitors of COX1 and COX2. Treatment with salubrinal, MG132 and COX2 inhibitor, like curcumin, prevented the replication of RSV and the epithelial responses, and treatment with salubrinal and MG132 enhanced the upregulation of tight junction molecules induced by infection with RSV. These results suggest that curcumin can prevent the replication of RSV and the epithelial responses to it without cytotoxicity and may act as therapy for severe lower respiratory tract disease in infants and young children caused by RSV infection.

Regulation of tight junctions in upper airway epithelium.

The mucosal barrier of the upper respiratory tract including the nasal cavity, which is the first site of exposure to inhaled antigens, plays an important role in host defense in terms of innate immunity and is regulated in large part by tight junctions of epithelial cells. Tight junction molecules are expressed in both M cells and dendritic cells as well as epithelial cells of upper airway. Various antigens are sampled, transported, and released to lymphocytes through the cells in nasal mucosa while they maintain the integrity of the barrier. Expression of tight junction molecules and the barrier function in normal human nasal epithelial cells (HNECs) are affected by various stimuli including growth factor, TLR ligand, and cytokine. In addition, epithelial-derived thymic stromal lymphopoietin (TSLP), which is a master switch for allergic inflammatory diseases including allergic rhinitis, enhances the barrier function together with an increase of tight junction molecules in HNECs. Furthermore, respiratory syncytial virus infection in HNECs in vitro induces expression of tight junction molecules and the barrier function together with proinflammatory cytokine release. This paper summarizes the recent progress in our understanding of the regulation of tight junctions in the upper airway epithelium under normal, allergic, and RSV-infected conditions.

Humulone suppresses replication of respiratory syncytial virus and release of IL-8 and RANTES in normal human nasal epithelial cells.

Respiratory syncytial virus (RSV) is the major infectious agent causing serious respiratory tract inflammation in infants and young children. However, an effective vaccine and anti-viral therapy for RSV infection have not yet been developed. Hop-derived bitter acids have potent pharmacological effects on inflammation. Therefore, we investigated the effects of humulone, which is the main constituent of hop bitter acids, on the replication of RSV and release of the proinflammatory cytokine IL-8 and chemokine RANTES in RSV-infected human nasal epithelial cells (HNECs). We found that humulone prevented the expression of RSV/G-protein, formation of virus filaments and release of IL-8 and RANTES in a dose-dependent manner in RSV-infected HNECs. These findings suggest that humulone has protective effects against the replication of RSV, the virus assembly and the inflammatory responses in HNECs and that it is a useful biological product for the prevention and therapy for RSV infection.

Hop water extract inhibits double-stranded RNA-induced thymic stromal lymphopoietin release from human nasal epithelial cells.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) acts as a master switch for allergic inflammation and plays a key role in allergic diseases, including allergic rhinitis. Double-stranded RNA (dsRNA) recognized by Toll-like receptor 3 (TLR3) strongly activates TSLP release from human nasal epithelial cells (HNECs). Hop (Humulus lupulus L.) extracts have been shown to have potent pharmacologic effects on inflammation. METHODS: To investigate whether a hop water extract (HWE) prevents TSLP release from HNECs, human telomerase reverse transcriptase (hTERT)-transfected HNECs, used as a model of normal HNECs, were pretreated with HWE before treatment with the TLR3 ligand Polyinosine-polycytidylic acid (poly[I:C]). RESULTS: In the hTERT-transfected HNECs, treatment with HWE significantly reduced poly(I:C)-induced production and release of TSLP in a dose-dependent manner, as well as dexamethasone. Treatment with the protein kinase C (PKC) inhibitor GF109203X and NF-κB inhibitor IMD-0354 also reduced poly(I:C)-induced TSLP release from hTERT-transfected HNECs. Treatment with HWE efficiently prevented up-regulation of PKC activity by 12-O-tetradecanoyl phorbol-13-acetate but not NF-κB activity induced by IL-1ß in hTERT-transfected HNECs. CONCLUSION: Our results clearly indicated that HWE inhibited dsRNA-induced production and release of TSLP via a PKC signal pathway in HNECs and it may have potent preventive effects against allergic rhinitis.

Mucosal immune barrier and antigen-presenting system in human nasal epithelial cells.

The upper respiratory tract including the nasal cavity, which is the first site of invading antigen exposure, plays a crucial role in host defense via the mucosal immune response. The epithelium of nasal mucosa forms a continuous barrier against a wide variety of exogenous antigens. The epithelial barrier function is regulated in large part by the apical-most intercellular junction, referred to as the tight junction. Antigen-presenting cells, particularly dendritic cells (DCs), are known to play an important role in human nasal mucosa. Recently, the author and colleagues discovered a new mechanism for pathogen uptake in the nasal mucosa, by which DCs open the tight junctions between epithelial cells and send dendrites outside the epithelium to directly sample the pathogen. In order to preserve the integrity of the epithelial barrier and penetrate beyond well-developed epithelial tight junctions, DCs express tight junction proteins. We also found that these DCs are activated by nasal epithelial-derived TSLP induced by stimuli such as cytokines and Toll-like receptor ligands. In this lecture, I will talk about the novel mechanisms in host defense in terms of innate immunity of the nasal mucosa from the point of view of the mucosal barrier function.

Respiratory syncytial virus infection and the tight junctions of nasal epithelial cells.

Respiratory syncytial virus (RSV) primarily infects upper respiratory tract cells, mainly nasal epithelial cells. The tight junctions of nasal epithelial cells are thought to perform important innate immune function against foreign materials including respiratory viruses. We investigated in vitro the relationship of RSV infection and the tight junctions of primary nasal epithelial cells which had been transfected with human telomerase reverse transcriptase (hTERT) to prolong cell life. Nasal epithelial cells developed tight junctions when cultured in medium containing fetal bovine serum, and these cells showed apparent resistance to RSV infection compared to control cells. RSV could infect these cells from apical but not basolateral side, suggesting that only apical side possess RSV receptor or a mechanism for absorbing RSV particles. Importantly, RSV infection of the cells enhanced the expression of tight junction proteins occludin, claudin-4 and ZO-1. These findings suggest that RSV infection induces polarity in the infected cells. This polarity could facilitate cellular secretion of propagated RSV, thereby spreading the infection.

Type-III interferon, not type-I, is the predominant interferon induced by respiratory viruses in nasal epithelial cells.

As an innate immune response against diverse viral infections, a host induces two types of interferon (IFN), type-I (IFN-ß/α) and type-III (IFN-λ). We investigated IFN inductions by respiratory viruses, including respiratory syncytial virus (RSV), measles virus and mumps virus in human nasal epithelial cells (NECs). IFN-λ, but not IFN-ß/α, was induced by respiratory virus infection in primary NECs and immortalized NECs through transfection with the human telomerase reverse transcriptase gene (hTERT-NECs). In contrast, both IFN-λ and IFN-ß/α were induced by RSV infection in human bronchiolar carcinoma cell line A549. Suppression of retinoic acid-inducible gene-I (RIG-I) expression using siRNA significantly reduced IFN-λ1 production in RSV-infected hTERT-NECs, while suppression of melanoma differentiation-associated gene 5 (MDA5) expression did not. Exogenous IFN-λ1 treatment suppressed RSV replication and chemokine induction in hTERT-NECs. These data indicate that IFN-λ, but not IFN-ß/α, contributes to the main first line defense via RIG-I-dependent pathway against respiratory virus infection in NECs.

A nuclear factor-κB signaling pathway via protein kinase C δ regulates replication of respiratory syncytial virus in polarized normal human nasal epithelial cells.

Respiratory syncytial virus (RSV) is the major cause of bronchitis, asthma, and severe lower respiratory tract disease in infants and young children. The airway epithelium, which has a well-developed barrier regulated by tight junctions, is the first line of defense during respiratory virus infection. In upper airway human nasal epithelial cells (HNECs), however, the primary site of RSV infection, the mechanisms of replication and budding of RSV, and the epithelial cell responses, including the tight junctional barrier, remain unknown. To investigate the detailed mechanisms of replication and budding of RSV in HNECs and the epithelial cell responses, we established an RSV-infected model using human telomerase reverse transcriptase--transfected HNECs. We first found that the expression and barrier function of tight junction molecules claudin-4 and occludin were markedly induced together with production of proinflammatory cytokines interleukin 8 and tumor necrosis factor-α in HNECs after RSV infection, and the induction of tight junction molecules possibly contributed to budding of RSV. Furthermore, the replication and budding of RSV and the epithelial cell responses in HNECs were regulated via a protein kinase C δ/hypoxia-inducible factor-1α/nuclear factor-κB pathway. The control of this pathway in HNECs may be useful not only for prevention of replication and budding of RSV, but also in therapy for RSV-induced respiratory pathogenesis.

Poly(I:C) reduces expression of JAM-A and induces secretion of IL-8 and TNF-α via distinct NF-κB pathways in human nasal epithelial cells.

Human nasal epithelium is an important physical barrier and innate immune defense protecting against inhaled substances and pathogens. Toll-like receptor (TLR) signaling, which plays a key role in the innate immune response, has not been well characterized in human nasal epithelial cells (HNECs), including the epithelial tight junctional barrier. In the present study, mRNAs of TLR1-10 were detected in hTERT-transfected HNECs, which can be used as an indispensable and stable model of normal HNECs, similar to primary cultured HNECs. To investigate the changes of tight junction proteins and the signal transduction pathways via TLRs in HNECs in vitro, hTERT-transfected HNECs were treated with TLR2 ligand P(3)CSK(4), TLR3 ligand poly(I:C), TLR4 ligand LPS, TLR7/8 ligand CL097, TLR8 ligand ssRNA40/LyoVec, and TLR9 ligand ODN2006. In hTERT-transfected HNECs, treatment with poly(I:C) significantly reduced expression of the tight junction protein JAM-A and induced secretion of proinflammatory cytokines IL-8 and TNF-α. Both the reduction of JAM-A expression and the induction of secretion of IL-8 and TNF-α after treatment with poly(I:C) were modulated by distinct signal transduction pathways via EGFR, PI3K, and p38 MAPK and finally regulated by a TLR3-mediated NF-κB pathway. The control of TLR3-mediated signaling pathways in HNECs may be important not only in infection by viral dsRNA but also in autoimmune diseases caused by endogenous dsRNA released from necrotic cells.

Epithelial barrier and antigen uptake in lymphoepithelium of human adenoids.

Invasion of antigens through the mucosal surface can be prevented by the common mucosal immune system, including Peyer's patches (PPs) and nasopharyngeal-associated lymphoreticular tissue (NALT). The adenoids (nasopharyngeal tonsils) comprise one of the NALTs and constitute the major part of Waldeyer's lymphoid ring in humans. However, the role of the lymphoepithelium, including M cells and dendritic cells (DCs), in the adenoids is unknown compared with the epithelium of PPs. NALTs also have unique functions such as the barrier of epithelial cells and uptake of antigens by M cells and DCs, and may play a crucial role in airway mucosal immune responses. The lymphoepithelium of adenoids has well-developed tight junctions that play an important role in the barrier function, the same as nasal epithelium but not palatine tonsillar epithelium. Tight junction molecules are expressed in both M cells and DCs as well as epithelial cells, and various antigens may be sampled, transported, and released to lymphocytes through the cells while they maintain the integrity of the epithelial barrier. This review summarizes the recent progress in our understanding of how M cells and DCs control the epithelial barrier in the adenoids.

c-Jun N-terminal kinase is largely involved in the regulation of tricellular tight junctions via tricellulin in human pancreatic duct epithelial cells.

Tricellulin (TRIC) is a tight junction protein at tricellular contacts where three epithelial cells meet, and it is required for the maintenance of the epithelial barrier. To investigate whether TRIC is regulated via a c-Jun N-terminal kinase (JNK) pathway, human pancreatic HPAC cells, highly expressed at tricellular contacts, were exposed to various stimuli such as the JNK activators anisomycin and 12-O-tetradecanoylphorbol 13-acetate (TPA), and the proinflammatory cytokines IL-1ß, TNFα, and IL-1α. TRIC expression and the barrier function were moderated by treatment with the JNK activator anisomycin, and suppressed not only by inhibitors of JNK and PKC but also by siRNAs of TRIC. TRIC expression was induced by treatment with the PKC activator TPA and proinflammatory cytokines IL-1ß, TNFα, and IL-1α, whereas the changes were inhibited by a JNK inhibitor. Furthermore, in normal human pancreatic duct epithelial cells using hTERT-transfected primary cultured cells, the responses of TRIC expression to the various stimuli were similar to those in HPAC cells. TRIC expression in tricellular tight junctions is strongly regulated together with the barrier function via the JNK transduction pathway. These findings suggest that JNK may be involved in the regulation of tricellular tight junctions including TRIC expression and the barrier function during normal remodeling of epithelial cells, and prevent disruption of the epithelial barrier in inflammation and other disorders in pancreatic duct epithelial cells.

PPARgamma agonists upregulate the barrier function of tight junctions via a PKC pathway in human nasal epithelial cells.

Peroxisome proliferator activated (PPAR)gamma plays a critical role in the control of not only adipocyte differentiation, lipid metabolism and immunity but also the barrier functions of epithelial and endothelial cells. In the present study, to investigate effects of PPAR gamma agonists on the tight junctional barrier of human nasal epithelial cells (HNECs), hTERT-transfected HNECs, which highly express both PPAR gamma and tight junction proteins, were treated with the PPAR gamma agonists rosiglitazone and troglitazone. Treatment with the PPAR gamma agonists enhanced the barrier function of hTERT-transfected HNECs together with the upregulation of tight junction molecules claudin-1 and -4, occludin, and tricellulin at the transcriptional level. A significant increase of tight junction strands was also observed after treatment with rosiglitazone. Treatment with PPAR gamma agonists induced the activity of phospho-PKC in hTERT-transfected HNECs. The upregulation of the tight junction molecules in hTERT-transfected HNECs by rosiglitazone was inhibited by not only PPAR gamma antagonists GW9662 and T0070907, but also the panPKC inhibitor GF109203X. These findings suggest that PPAR gamma agonists upregulate the barrier function of tight junctions of human nasal epithelial cells via a PKC signaling pathway and could be novel drugs for protection against inhaled substances and pathogens in the airway epithelium of human nasal mucosa.

Expression and localization of tricellulin in human nasal epithelial cells in vivo and in vitro.

Tricellulin was identified as the first marker of the tricellular tight junction, which forms at the meeting points of three cells, and it is required for the maintenance of the transepithelial barrier. Although it is also considered to be important for the mucosal barrier of the upper respiratory tract, little is known about its expression and localization. In the present study, we examined the expression and localization of tricellulin in normal human nasal epithelial cells in vivo and in vitro, especially using primary cultures and telomerase reverse transcriptase (hTERT)-transfected cells. In human nasal epithelial cells in vivo and in vitro, mRNA and protein of tricellulin were detected. It was localized not only at tricellular contacts but also at bicellular borders, and in part colocalized with occludin. In human nasal epithelium, by immunoelectron microscopy analysis, tricellulin-associated gold particles were observed around the junction-like structure of the uppermost region. By treatment with 10% fetal bovine serum (FBS), expression of tricellulin mRNA was weakly increased, whereas that of bicellular tight junction molecules was strongly increased, in real-time PCR. These results suggest that tricellulin is stably expressed in human nasal epithelial cells and may play an important role for the sealing of the corner at tricellular contacts to prevent infiltration by various inhaled viruses and antigens.

Induction of JAM-A during differentiation of human THP-1 dendritic cells.

Junctional adhesion molecule (JAM)-A is not only localized at tight junctions of endothelial and epithelial cells but is also expressed on circulating leukocytes and dendritic cells (DCs). In the present study, to investigate the regulation of JAM-A in DCs, mature DCs were differentiated from the human monocytic cell THP-1 by treatment with IL-4, GM-CSF, TNF-alpha, and ionomycin, and some cells were pretreated with the PPAR-gamma agonists. In the THP-1 monocytes, mRNAs of tight junction molecules, occludin, tricellulin, JAM-A, ZO-1, ZO-2 and claudin-4, -7, -8, and -9 were detected by RT-PCR. In mature DCs that had elongated dendrites, mRNA and protein of JAM-A were significantly increased compared to the monocytes. PPAR-gamma agonists prevented the elongation of dentrites but not upregulation of JAM-A in mature DCs. These findings indicated that the induction of JAM-A occurred during differentiation of human THP-1 DCs and was independent of PPAR-gamma and the p38 MAPK pathway.