bluuebunny/crossref_metadata_2025 huggingface dataset:
The ichq mutant mouse, a model for the human skin disorder harlequin ichthyosis: mapping, keratinocyte culture, and consideration of candidate genes involved in epidermal growth regulation
Authors: Dunnwald Martine , Zuberi Aamir R. , Stephens Karen , Le Rosalynda , Sundberg John P. , Fleckman Philip , Dale Beverly A.
Published: June 2003

Abstract: Harlequin ichthyosis (HI) is a rare and usually fatal scaling skin disorder. The HI mutant mouse (ichq/ichq) has many similarities to the human disorder and provides an important model to identify candidate genes. In this study, we report refined mapping of the mouse ichq locus and consideration of the candidate genes: calpain 1 (Capn1), phospholipase C beta 3 (Plcb3), and Rela and Ikka/Chuk that encode components of the nuclear factor‐kappa B (NF‐κB) pathway. Each are strong candidates because of epidermal expression and/or changes in expression in human HI. All candidates are linked to the ichq locus on mouse Chromosome 19, although Ikka is located more distally. Genetic mapping in mouse has narrowed the ichq critical region to 4 cM. Keratinocytes from skin of +/+, +/ichq and ichq/ichq mice were cultured; all genotypes had similar expression of epidermal differentiation markers. RT‐PCR amplification and sequence analysis of each candidate gene did not reveal any mutations in the ichq mouse. Mutational screening of CAPN1 cDNA from different human HI cases revealed a R433P change, but analysis of 50 normal samples demonstrated that this was an apparent polymorphism. Sequence of RELA in five unrelated human HI cases was normal. The results provide compelling evidence that none of these genes are the primary defect in the ichq mouse and that CAPN1 and RELA are not mutated in the human disorder.
Immunomapping of EBA sera to multiple epitopes on collagen VII: further evidence that anchoring fibrils originate and terminate in the lamina densa
Authors: McMillan James R. , Matsumura Tetsuri , Hashimoto Takashi , Schumann Hans , Bruckner‐Tuderman Leena , Shimizu Hiroshi
Published: June 2003

Abstract: Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease with circulating antibodies to type VII collagen, a major component of anchoring fibrils located at the dermal‐epidermal junction. The purpose of this study was to further confirm the ultrastructural organisation of anchoring fibrils and to assess the relationship between the clinical phenotype of EBA and target site of their autoantibodies on anchoring fibrils. We studied the ultrastructural binding site of circulating autoantibodies from two patients with atypical clinical features who predominantly presented with oral lesions, and compared this with two patients with clinically typical forms of EBA. Immunoblotting of whole dermal extracts showed labelling of 290‐kDa bands consistent with that of type VII collagen as well as the non‐collagenous (NC‐1) domain fusion protein in three out of four patients' sera. Postembedding immunoelectron microscopy (IEM) using Lowicryl K11M embedded normal human skin and patients' sera demonstrated the majority of labelling within the lamina densa, not below the lamina densa. We conclude that EBA autoantibodies in these patient's sera bind to the NC‐1 domain of collagen VII situated in the lamina densa of the epidermal basement membrane, regardless of the EBA clinical phenotype. This confirms the previous notion that anchoring fibrils originate and terminate in the lamina densa.
UVB‐induced inflammation gives increased d‐dopachrome tautomerase activity in blister fluid which correlates with macrophage migration inhibitory factor
Authors: Sonesson Björn , Rosengren Evald , Hansson Ann‐Sofie , Hansson Christer
Published: June 2003

Abstract: UVB light was used to induce an experimental inflammation in normal human skin in order to investigate its correlation with the activity of the newly described enzyme d‐dopachrome tautomerase (DDT) in the fluid of experimental blisters. Macrophage migration inhibitory factor (MIF) activity was determined as a closely related marker of inflammation. DDT and MIF activities were demonstrated in blister fluids in all 10 healthy subjects. All but one of these subjects showed increased activity of DDT and MIF after three minimal erythemal doses (MED) of UVB. The mean activity of DDT increased approximately twofold and the mean activity of MIF also increased twofold after UVB in our experimental model. We found a strong correlation between DDT and MIF activities. The presence of DDT in epidermis and its increase at UV irradiation was confirmed by immunohistochemical studies. In this study, DDT is for the first time demonstrated in the skin. It is also the first time DDT can be related to inflammation, and its covariation with MIF strengthens this observation.
Expression of claudin‐5 in dermal vascular endothelia
Authors: Morita Kazumasa , Sasaki Hiroyuki , Furuse Kyoko , Furuse Mikio , Tsukita Shoichiro , Miyachi Yoshiki
Published: June 2003

Abstract: Claudins and occludin are integral membrane proteins at tight junctions (TJs). We examined subcellular localization of claudin‐5 and occludin in dermal vascular endothelia. Immunofluorescence staining showed that claudin‐5 was expressed at the cell–cell border of dermal vascular endothelia in mouse skin. However, in some dermal vessels, claudin‐5 expression was markedly decreased or absent in amount by double‐immunofluorescence stainings with PECAM‐1 and PAL‐E. In contrast, occludin was not detected in dermal vessels. Freeze‐fracture and immunoreplica electron microscopy on primary‐cultured human dermal endothelial cells showed that claudin‐5 was localized at tight junctions. These findings confirmed that TJs in dermal vascular endothelial cells are composed of claudin‐5.
Fibroblast growth factor‐2 but not Mel‐CAM and/or β3 integrin promotes progression of melanocytes to melanoma
Authors: Meier Friedegund , Caroli Ulrich , Satyamoorthy Kapaettu , Schittek Birgit , Bauer Jürgen , Berking Carola , Möller Helga , Maczey Evelyn , Rassner Gernot , Herlyn Meenhard , Garbe Claus
Published: June 2003

Abstract: A variety of melanoma‐associated antigens have been identified that mediate adhesion, growth, proteolysis, and modulation of immune response. However, the mechanisms by which human normal melanocytes become malignant are not clearly understood. Among the most consistent observations is the up‐regulation of fibroblast growth factor‐2 (FGF‐2) and of the adhesion molecules β3 integrin and Mel‐CAM during melanoma progression. To evaluate the potential role of FGF‐2, β3 integrin and Mel‐CAM in melanoma development we overexpressed FGF‐2, β3 integrin and Mel‐CAM in normal human melanocytes using replication‐deficient adenoviruses as a gene delivery vehicle. Fibroblast growth factor‐2 overexpressing melanocytes in monolayer culture displayed cytological atypia. Furthermore, in human skin reconstructs where the physiological milieu is recreated in vitro, FGF‐2‐overexpressing melanocytes exhibited marked proliferation, upwards migration, cluster formation and type IV collagen expression within the epidermal compartment, simulating early radial growth phase melanoma. In contrast, overexpression of β3 integrin and/or Mel‐CAM in melanocytes did not affect their biological behaviour in human skin reconstructs. The described results of the current and previous studies emphasise the key role of FGF‐2 in melanoma development and progression, underscoring the promise of FGF‐2 as a target for therapy.
Production of a monoclonal antibody, DF‐5, that identifies cells at the epithelial–mesenchymal interface in normal human skin. APN/CD13 is an epithelial–mesenchymal marker in skin
Authors: Sorrell J. Michael , Baber Marilyn A. , Brinon Laure , Carrino David A. , Seavolt Maralyn , Asselineau Daniel , Caplan Arnold I.
Published: June 2003

Abstract: Epithelial–mesenchymal interactions play a critical role in skin development and differentiation, and similar interactions may also regulate the day‐to‐day proliferation and differentiation events of the epidermis that occur in normal adult skin. This study was directed at identifying molecules that are selectively located at the dermal–epidermal junction in normal adult skin as they may be involved in regulating these homeostatic events. To this end, monoclonal antibodies were raised against the crude cell membrane fraction of cultured human dermal fibroblasts. Screening of antibodies that recognized cell surface antigen on cultured human dermal fibroblasts was followed by determining which of these antibodies selectively localized cells at sites of epithelial–mesenchymal interactions. Antibody DF‐5 fit these criteria and was further characterized. This antibody was found to recognize the cell surface ectopeptidase aminopeptidase N (APN), a molecule homologous to the cluster differentiation antigen CD13. Antibody DF‐5 and anti‐CD13 antibodies both identified cells at sites of epithelial–mesenchymal interactions in fetal, neonatal, and adult human skin, and the APN/CD13 enzyme activity was also identified at these sites. A second ectopeptidase, dipeptidyl peptidase IV (DPPIV) or CD26, presented a significantly different immunohistochemical and histochemical pattern in skin samples, confirming the specificity of the APN/CD13 studies. The function of APN/CD13 in skin has yet to be determined. Its invariant localization at sites of epithelial–mesenchymal interactions argues for a role particular to this region. It may play a role in regulating the activity of neuropeptides or other signaling peptides that are released in this region of skin or it may have an as yet undefined role in mediating communication between dermal and epidermal cells.
Granulocyte colony stimulation factor (G‐CSF) suppresses interleukin (IL)‐12 and/or IL‐2 induced interferon (IFN)‐γproduction and cytotoxicity of decidual mononuclear cells
Authors: Sugita Kenji , Hayakawa Satoshi , Karasaki‐Suzuki Miki , Hagiwara Hiroshi , Chishima Fumihisa , Aleemuzaman Sheikh , Li Jong‐a , Nishinarita Susumu , Yamamoto Tatsuo
Published: June 2003

Problem: The placenta is one of the few non‐hematopoietic tissues to express granulocyte colony stimulation factor (G‐CSF). Placental G‐CSF production is considered to be one of the major causes of granulocytosis during pregnancy although its physiological role in pregnancy has not yet been examined.

Method of study: The effects of G‐CSF on interleukin (IL)‐2 and/or IL‐12 induced interferon (IFN)‐γ production of magnetic cell sorting (MACS) sorted decidual lymphocytes was examined by enzyme‐linked immunosorbent spot‐forming cell assay (ELISPOT). The effect of G‐CSF on cytotoxicity of decidual lymphocytes against the choriocarcinoma cell line JEG‐3 was examined by lactate dehydrogenase (LDH) release assay.

Results: As previously reported by us, IL‐2 and/or IL‐12 activated decidual mononuclear cells were capable of killing choriocarcinoma cells. We observed that G‐CSF abolished IFN‐γ production and cytotoxicity of decidual mononuclear cells and MACS sorted CD56+ cells.

Conclusions: In addition to its well‐known trophic effects on hematopoiesis, our results suggest about new roles of G‐CSF in reproductive immunology.
Concentrations of Interferon‐γ‐Induced Protein‐10 (IP‐10), an Antiangiogenic Substance, are Decreased in Peritoneal Fluid of Women with Advanced Endometriosis
Authors: Yoshino Osamu , Osuga Yutaka , Koga Kaori , Hirota Yasushi , Tsutsumi Osamu , Yano Tetsu , Morita Yutaka , Momoeda Mikio , Fujiwara Toshihiro , Kugu Koji , Taketani Yuji
Published: June 2003

Problem:  To assess whether interferon‐γ‐induced protein‐10 (IP‐10), a chemokine that has antiangiogenic activities, may be involved in the pathogenesis of endometriosis.

Method of Study:  A total of 120 patients undergoing laparoscopy for pain and/or infertility were recruited, and peritoneal fluid (PF) and bone marrow derived cells in PF were collected. Concentrations of IP‐10 in PF were measured with a specific enzyme‐linked immunosorbent assay. Expression of IP‐10 and IP‐10 receptor, CXCR3, in bone marrow derived cells in PF, peritoneum and endometriotic cells was analyzed by reverse transcription‐polymerase chain reaction.

Results:  All of the PF samples examined contained detectable concentrations of IP‐10. In women with advanced stages of endometriosis, IP‐10 concentrations in PF were significantly lower than those of women in early stages (P = 0.02). The IP‐10 concentrations in women with advanced endometriosis also appeared to be lower than those without endometriosis although the difference was statistically marginal (P = 0.06). The expression of both IP‐10 and CXCR3 was clearly detected in the bone marrow derived cells in PF, peritoneum and endometriotic stromal cells.

Conclusions:  Decreased concentrations of IP‐10 in PF from women with advanced stages of endometriosis may imply that the peritoneal environment of these women is permissive to the development of the disease by enhancing angiogenesis and/or modulating inflammatory/immunological responses.
Immunology and Endometriosis
Authors: Berkkanoglu Murat , Arici Aydin
Published: June 2003

Problem: Accumulating data suggests that aberrant immune responses during retrograde menstruation may be involved in the development of endometriosis.

Method of Study: The role of immunology in the etiology of endometriosis is reviewed and summarized from the available literature.

Results: Immunologic factors may affect a woman's susceptibility to implantation of exfoliated endometrial cells. Immune alterations include increased number and activation of peritoneal macrophages, decreased T cell reactivity and natural killer cell cytotoxicity, increased circulating antibodies, and changes in the cytokine network.

Conclusion: There is substantial evidence that immunologic factors play a role in the pathogenesis of endometriosis and endometriosis‐associated infertility. Decreased natural killer cell cytotoxicity leads to an increased likelihood of implantation of endometriotic tissue. In addition, macrophages and a complex network of locally produced cytokines modulate the growth and inflammatory behavior of ectopic endometrial implants.
Butyrylcholinesterase Activity and Pregnancy‐Associated Differences in Immunologically Relevant Peripheral Blood Leukocyte Populations
Authors: Mahmoud Fadia , Haines David , Abul Habib , Omu Alexander
Published: June 2003

Problem:  Toxic anticholinesterases (AC) are known contributors to negative pregnancy outcome. Impairment of detoxification mechanisms may correlate with occurrence of pregnancy disorders in Kuwait.

Method of Study:  Butyrylcholinesterase (BuChE), an enzyme which detoxifies AC was evaluated in 18 Kuwaiti women with pregnancy‐induced hypertension (PIH), compared with 15 healthy pregnant and eight healthy non‐pregnant women. T‐lymphocyte subpopulations were measured by flow cytometry, and BuChE activity was measured by spectrophotometry.

Results:  Unlike the PIH group, the normal pregnancy group exhibited a significant increase in BuChE activity compared with non‐pregnant control subjects (P = 0.04). Within the PIH cohort, inverse correlations were observed between BuChE activity and percentage of CD4+ CD25+ cells (P = 0.001), and CD8+ CD25+ cells (P = 0.007).

Conclusions:  Elevated BuChE activity in normal pregnancy may correlate with better ability to clear pregnancy‐threatening toxins, while lesser ability to do this in PIH women may be a contributor to disease. The fact that PIH subjects with large subpopulations of activated T cells also exhibited low BuChE activity further suggests a correlation between susceptibility to pregnancy loss and decreased activity of the enzyme.
Distribution of Immunocompetent Cells in Decidua of Controlled and Uncontrolled (Choriocarcinoma/Hydatidiform mole) Trophoblast Invasion
Authors: Knoeller S. , Lim E. , Aleta L. , Hertwig K. , Dudenhausen J. W. , Arck P. C.
Published: June 2003

Problem:  Pregnancy has been considered as a model of successfully controlled tissue invasion where trophoblast cells infiltrate the maternal decidua without being rejected or without destroying the tissue. In choriocarcinoma (CC) and hydatidiform mole (HM), a dysregulation of invasive (malignant/benign) trophoblast cells is present. Immunocompetent cells (IC) are known to be involved in rejection pathways of malignant cells and can also be identified in early pregnancy decidua. The aim of the present study was to identify the phenotype of IC in decidua of women with normal pregnancy (NP), CC and HM.

Methods:  Immunocompetent cells were detected by immunohistochemistry in decidual tissue from first trimester NP (n = 10), CC (n = 12) and HM (n = 11) using antibodies against CD8+, CD3+, CD56+, CD68+ cell surface markers and mast cell tryptase (MCT). A scaled eye piece was used for cell counting to obtain semiquantitative results. Statistical analysis was performed using Wilcoxon rank/Mann–Whitney tests.

Results:  We observed a significantly increased number of lymphocytes positive for CD8, CD3 and MCT positive granulocytes in CC and HM compared with the samples from NP (all P ≤ 0.001). Lymphocytes positive for natural killer (NK) cell marker CD56 were significantly decreased in CC and HM versus NP (P ≤ 0.001). The number of CD68 positive cells (macrophages) were not significantly different among the tissue pools.

Conclusion:  The increase of CD8/CD3 T cells and mast cells in CC and HM and the decrease of CD56 cells, compared with NP, suggests the necessity of a balance between T and NK cells in controlling trophoblast invasion.
Heme Oxygenases in Pregnancy II: HO‐2 is Downregulated in Human Pathologic Pregnancies
Authors: Zenclussen A. C. , Lim E. , Knoeller S. , Knackstedt M. , Hertwig K. , Hagen E. , Klapp B. F. , Arck P. C.
Published: June 2003

Problem:  We previously reported a diminished expression of the heme‐degrading enzymes heme oxygenases (HO)‐1 and HO‐2 in decidua and placenta from mice undergoing Th1‐mediated abortion, strongly indicating the protective effect of HO in murine pregnancy maintenance. Here we investigated whether the expression of HO‐1 and HO‐2 is also reduced at the feto‐maternal interface of pathologic human pregnancies.

Method of Study:  Immunohistochemistry was used to detect HOs expression in placental and decidual first‐trimester tissue from patients with: spontaneous abortion (n = 14), choriocarcinoma (n = 14), hydatidiform mole (H‐mole) (n = 12), compared with normally progressing pregnancies (n = 15). Further, we investigated early third‐trimester decidual and placental tissue from patients with pre‐eclampsia (n = 13) compared with fetal growth retardation (n = 14) as age‐matched controls.

Results:  In first trimester tissue, we observed a significant reduction of HO‐2 expression in invasive trophoblast cells, endothelial cells, and syncytiotrophoblasts in samples from patients with spontaneous abortion compared with normal pregnancy. H‐mole samples showed a diminished expression of HO‐2 in invasive trophoblast cells and endothelial cells in comparison with NP, whereas choriocarcinoma samples showed no significant differences compared with the control. In third trimester tissue, HO‐2 was also reduced in syncytiotrophoblasts and invasive trophoblast cells from pre‐eclampsia compared with samples from fetal growth retardation. HO‐1 expression was diminished in all pathologies investigated; however, the differences did not reach levels of significance.

Conclusions:  Our data indicate that HOs play a crucial role in pregnancy and low expression of HO‐2, as observed in pathologic pregnancies, may lead to enhanced levels of free heme at the feto‐maternal interface, with subsequent upregulation of adhesion molecules, allowing enhanced inflammatory cells migration to the feto‐maternal interface.
Inherited Thrombophilia: Impact on Human Reproduction
Authors: Buchholz Tina , Thaler Christian J.
Published: June 2003

The development of thrombotic disorders is a major threat for young women during pregnancy. It is one of the main causes of pregnancy‐related disorders, which may also result in harm for the conceptus. Successful pregnancies require an even balance of coagulation and fibrinolysis, in order to secure stabilization of the basal plate as well as adequate placental perfusion. Thrombophilia is a laboratory definition for pre‐disposing factors of thrombosis, which can be inherited or acquired. Many individuals, who carry a thrombotic defect remain asymptomatic, at least until additional boosting factors arise. The documentation of thrombophilic causes of recurrent miscarriage or pregnancy‐related disorders is important, because of availability of effective early treatment. There is a rapidly growing awareness on the relationship of genetic factors influencing hemostasis and pregnancy‐related disorders. The aim of our review is to summarize this knowledge, focusing on common genetic variations.
Antinuclear Autoantibodies, Complement Level, Hypergammaglobulinemia and Spontaneous Intrauterine Hematoma in Pregnant Women
Authors: Alijotas Jaume , Izquierdo Maximo , Serra Bernat , Cusido Ma Teresa , Ribera Miquel , Carrera Josep Ma
Published: June 2003

Problem: To examine the associative relationship among autoantibodies, C4 levels and intrauterine hematomas (IUH) in more detail than in the studies published earlier.

Method Of Study: We performed a retrospective study of 54 women with poor obstetric outcomes. Sera were screened for antinuclear antibodies (ANA), anti‐DNA antibodies, antiphospholipid antibodies (aPL), and antithyroid antibodies. C4‐complement and gammaglobulin levels were also monitored. We compared the main variables in IUH complicated pregnancy group with the risk pregnancy group without IUH. We also compared these variables in the IUH cases before and during IUH.

Results: Eight IUH were detected. The average number of spontaneous losses for these eight women was 3.3 ± 2.1 (range: 1–8). aPL was present in 100% of cases. ANAs and hypergammaglobulinemia were present in 50% of cases and low C4 in 87.5% of cases. After comparing these variables apart from C4 before and during IUH, we found no statistical differences. However, C4 was low in four patients before IUH and in seven patients during IUH (OR: 7.0; 95% CI: 0.57–86.33). When we compared autoantibodies apart from lupus anticoagulant (LAC) between the two groups, no differences were observed. However, seven of the eight (87.5%) patients with IUH were LAC positive whereas only 24 of the 46 patients (52.1%) were positive in the non‐IUH group (OR: 6.42; 95% CI: 0.73–56.41). Rapid plasma reagin was present in 8/46 in the non‐IUH group (16.7%) and 5/8 in the IUH group (62.5%) P < 0.015).

Conclusions: In women with poor obstetric histories, autoantibodies, especially antiphospholid antibodies, may play a role in the IUH development especially if low C4 and/or hypergammaglobulinemia are present.
Stimulatory Effect of the Herbal Medicine Keishi‐bukuryo‐gan on a Cytokine‐induced Neutrophil Chemoattractant, in Rat Ovarian Cell Culture
Authors: Yasui Toshiyuki , Matsuzaki Toshiya , Ushigoe Kenjiro , Kuwahara Akira , Maegawa Masahiko , Furumoto Hiroyuki , Aono Toshihiro , Irahara Minoru
Published: June 2003

Problem and Method of Study:  We investigated the effects of Keishi‐bukuryo‐gan, a Japanese herbal medicine, and its crude ingredients in relation to the production of cytokine‐induced neutrophil chemoattractant (CINC/gro), interleukin 1β (IL‐1β) and tumor necrosis factor α (TNFα), which are known to stimulate the secretion of CINC/gro in the ovulatory process, and the effects of Keishi‐bukuryo‐gan with those of Toki‐shakuyaku‐san, which has been shown to have an effect on the ovary. We cultured whole ovarian dispersates from immature (3‐week‐old) female rats with Keishi‐bukuryo‐gan, Toki‐shakuyaku‐san and crude ingredients of Keishi‐bukuryo‐gan. The contents of CINC/gro, IL‐1β and TNFα in the cultured media were determined by enzyme‐linked immunosorbent assay (ELISA).

Results:  Keishi‐bukuryo‐gan stimulated the secretion of CINC/gro in a dose‐dependent manner, and the secretion of CINC/gro into the culture medium increased significantly at concentrations of Keishi‐bukuryo‐gan of 10 and 100 μg/mL (P < 0.001). The stimulatory effect of Keishi‐bukuryo‐gan on the production of CINC/gro is significantly (P < 0.001) stronger than that of Toki‐shakuyaku‐san at the same concentrations of 100 μg/mL. In addition, Keishi‐bukuryo‐gan stimulated the secretion of IL‐1β in a dose‐dependent manner, while it did not stimulate the secretion of TNFα even at a concentration of 100 μg/mL. Moutan Cortex, Paeoniae Radix and Persicae Semen, which are crude ingredients of Keishi‐bukuryo‐gan, enhanced the secretion of CINC/gro significantly (P < 0.01) in cultured whole ovarian dispersates.

Conclusions:  These results show that Keishi‐bukuryo‐gan can stimulate the secretion of CINC/gro as well as the production of IL‐1β and that this stimulatory effect of Keishi‐bukuryo‐gan was significantly stronger than that of Toki‐shakuyaku‐san in immature rat ovarian cell culture.
Possible Modulators of IL‐8 and GRO‐α Production by Granulosa Cells
Authors: Zeineh Kosay , Kawano Yasushi , Fukuda Junichiro , Nasu Kaei , Narahara Hisashi , Miyakawa Isao
Published: June 2003

Problem:  Interleukin‐8 (IL‐8) and growth‐regulated oncogene‐α (GRO‐α) have been proved to be important modulators of leukocyte chemotaxis in the mechanism of human ovulation. This study investigated the possible effects of IL‐1α, tumor necrosis factor‐α (TNF‐α), protein kinase C (PKC) activators (TPA), and db‐cyclic adenosine monophosphate (cAMP) on IL‐8 and GRO‐α production by immortalized GC1a and granulosa‐lutein cells.

Method of Study:  Confluent granulosa‐lutein cells were placed in serum‐free medium before incubated for 8 hr with the above‐mentioned test agents. Finally, we measured IL‐8 and GRO‐α levels in the culture media using an enzyme‐linked immunosorbent assay (ELISA).

Results:  Treatment of granulosa‐lutein cells with of IL‐1α (1 nM), TNF‐α (1 nM), TPA (1 nM) and db‐cAMP (100 μM) produced higher levels of IL‐8 than untreated cells by 8 hr (2274.7 ± 146.3, 1489.8 ± 190.1, 1452.9 ± 152.7, 1313.6.6 ± 48.4 pg/mL, respectively; control = 457.7 ± 38.2 pg/mL; P < 0.001). Treatment of granulosa‐lutein cells with 1 nM of IL‐1α, TNFα, TPA, and db‐cAMP (100 μM) resulted in higher levels of GRO‐α than untreated cells by 8 hr (993.7 ± 9.5, 171.4 ± 6.5, 147.5 ± 6.7, 472.4 ± 16.2 pg/mL respectively; control = 73.8 ± 8.2 pg/mL; P < 0.001).

Conclusions:  Our data strongly suggests roles for IL‐1α, TNFα, and PKC activators in the inflammation–like mechanism of human ovulation. Furthermore, our study suggests a positive, but still debatable, role for cAMP in the same mechanism.
Deficient Expression of Tumor Necrosis Factor Receptor Type 2 in the Endometrium of Women with Endometriosis
Authors: Kharfi Abdelaziz , Labelle Yves , Mailloux Jacques , Akoum Ali
Published: June 2003

Problem: Tumor necrosis factor‐α (TNF‐α) is secreted mainly during the menstrual phase and has been suggested to play a role in induction of apoptosis in endometrial cells and menstrual shedding. TNF‐α receptor type 2 (TNF‐RII) is believed to play a central role in TNFα‐mediated cytotoxic, mitogenic, anti‐proliferative and apoptotic effects. The aim of this study was to assess whether TNF‐RII maybe expressed differentially in the endometrium of women with different degrees of endometriosis.

Method of study: TNF‐RII expression in the endometrial tissue of women with and without endometriosis was investigated by immunohistochemical techniques and in situ hybridization.

Results: In histological sections, we observed TNF‐RII mRNA and the corresponding protein localized mainly in endometrial glandular cells, with only very faint immunostaining in the surrounding stromal cells. Statistical analysis of our data showed a significant decrease in protein and mRNA expression of TNF‐RII in endometrial glandular cells of patients with stages I and II endometriosis compared to normal subjects. TNF‐RII expression was also found to decrease significantly in the secretory phase of the menstrual cycle in women with early endometriosis stages (I and II).

Conclusions: In view of the relevant role of TNF‐RII in the modulation of the inflammatory and the proapoptotic effects of TNFα, deficient expression of TNF‐RII mRNA in the endometrium of women at the earliest stages of endometriosis may play a significant role in the pathophysiology of this disease.
Expression of Allograft Inflammatory Factor‐1 in Mouse Uterus and Poly (I:C)‐induced Fetal Resorption
Authors: Shimada Shigeki , Iwabuchi Kazuya , Watano Keiko , Shimizu Hidemi , Yamada Hideto , Minakami Hisanori , Onoé Kazunori
Published: June 2003

Problem: To investigate whether the allograft inflammatory factor‐1 (AIF‐1) is expressed and plays a role in the reproductive system.

Method of study: AIF‐1 expression was examined in uteri of non‐pregnant and pregnant mice by Northern blot analysis, RT‐PCR and immunohistochemistry.

Results: The expression of AIF‐1 varied during the estrous cycle with a peak at estrus. After the insemination, the expression of AIF‐1 mRNA diminished gradually and again increased in the pre‐implantation or implantation period in allogeneic or syngeneic pregnancy, respectively. Enhanced expressions of AIF‐1, tumor necrosis factor‐α (TNF‐α) and nitric oxide synthase 2 (NOS2) mRNA were observed in the embryos of resorption‐prone pregnancy injected with poly(I:C).

Conclusions:  This study demonstrated for the first time that AIF‐1 was expressed in uterus. The expression level was associated with the population size of macrophage and varied during the estrous cycle and the pregnancy period. The augmented expression of AIF‐1 with concomitant expressions of TNF‐α and NOS2 mRNA in poly(I:C)‐injected mice suggests a correlation between AIF‐1 production and fetal resorption.
Relationships between Chlamydia trachomatis Antibody Titers and Tubal Pathology Assessed using Transvaginal Hydrolaparoscopy in Infertile Women
Authors: Shibahara Hiroaki , Takamizawa Satoru , Hirano Yuki , Ayustawati  , Takei Yuji , Fujiwara Hiroyuki , Tamada Saori , Sato Ikuo
Published: June 2003

Problem: Since transvaginal hydrolaparoscopy (THL) was introduced as the first‐line procedure in the early stages of the exploration of the adnexal structures in infertile women, it has been shown that THL is a less traumatic and a more suitable outpatient procedure than diagnostic laparoscopy. This study was performed to investigate the relationships between Chlamydia trachomatis antibody titers and tubal pathology assessed using THL in infertile women.

Methods: The C. trachomatis antibody titers (IgG and IgA) were evaluated by ELISA. The posterior of the uterus and the tubo‐ovarian structures were carefully observed, and tubal passage using indigocarmine was confirmed using THL. THL was carried out in 32 infertile women having C. trachomatis antibody in their sera between May 1999 and October 2001. Unilateral salpingectomy had been performed on two of the 32 patients.

Results: Tubal occlusion was confirmed in 20 (32.3%) of the 62 tubes, while peritubal adhesion was diagnosed in 37 (59.7%) of the 62 tubes. Using receiver operating characteristics curves, the cut‐off value of C. trachomatis IgG antibody titer to predict tubal occlusion was determined to be 3.55. Tubal occlusion was observed in 16 (51.6%) of the 31 tubes in patients with the C. trachomatis IgG antibody titer of more than 3.55, which was significantly higher in four (12.9%) of the 31 tubes having the antibody titer less than 3.55 (P = 0.004). However, there was no correlation between C. trachomatis IgG antibody titer and peritubal adhesion. As for C. trachomatis IgA antibody titer, there was no correlation between antibody titer and tubal occlusion or peritubal adhesion.

Conclusions: These results suggest that C. trachomatis infection is significantly associated with tubal pathology. Although the cut‐off value of C. trachomatis IgG antibody titer to predict the existence of tubal occlusion was shown to be 3.55, we would suggest that THL or standard laparoscopy is performed to consider appropriate treatments in patients with past C. trachomatis infection because of the high prevalence of peritubal adhesion.
An Immunohistochemical Study of the Secretory Immune System in Human Fetal Membranes and Decidua of the First Trimester of Pregnancy
Authors: Gurevich Pavel , Elhayany Asher , Ben‐Hur Herzl , Moldavsky Moisey , Szvalb Sergio , Zandbank Judit , Shperling Isai , Zusman Itshak
Published: June 2003

Problem: We analyzed the presence and distribution of components of the secretory immune system (SIS) in human fetal membranes (amnion, yolk sac, chorion) and decidua from the first trimester of pregnancy.

Materials and methods: Specimens from 17 embryos (4–8 weeks of pregnancy) and nine fetuses (9–12 weeks) were divided into those that had not been exposed to massive foreign antigenic effects (Group I, n = 18) and those that had suffered acute chorioamnionitis (Group II, n = 8).

Results: Positive immunostaining for secretory component (SC), joining (J) chain, IgG, IgA, and macrophages was seen from 4 to 5 weeks of development and then during the whole first trimester of pregnancy in the syncytio‐and cytotrophoblasts, the amniotic epithelium, the yolk sac endoderm and decidual cells. Macrophages with J chain, IgG and IgA were found in embryonic tissues on week 4, whereas lymphocytes, including those synthesizing IgA and IgM, appeared only at the end of the first trimester of pregnancy. In the decidua, lymphocytes and macrophages were recognized during the whole period of study. In cases with chorioamnionitis (Group II), reactivity of IgG and IgA in the mentioned cells of fetuses decreased sharply while the rate of immunoreactivity of SC and J chain as well as the number of T and B lymphocytes did not change. In the decidua, the number of immune reactive cells sharply increased with the appearance of plasma cells.

Conclusions: In the fetal membranes and decidua all the SIS components are present. We suggest that two SIS, maternal and fetal, participate in the formation of the barrier between a mother and the fetus. Both these systems have different origin and cellular content as well as different immune reactions.
SB‐656104‐A, a novel selective 5‐HT7 receptor antagonist, modulates REM sleep in rats
Authors: Thomas David R , Melotto Sergio , Massagrande Mario , Gribble Andrew D , Jeffrey Phillip , Stevens Alexander J , Deeks Nigel J , Eddershaw Peter J , Fenwick Susan H , Riley Graham , Stean Tania , Scott Claire M , Hill Matthew J , Middlemiss Derek N , Hagan Jim J , Price Gary W , Forbes Ian T
Published: June 2003

(6‐((R)‐2‐{2‐[4‐(4‐Chloro‐phenoxy)‐piperidin‐1‐yl]‐ethyl}‐pyrrolidine‐1‐sulphonyl)‐1H‐indole hydrochloride) (SB‐656104‐A), a novel 5‐hydroxytryptamine (5‐HT7) receptor antagonist, potently inhibited [3H]‐SB‐269970 binding to the human cloned 5‐HT7(a) (pKi 8.7±0.1) and 5‐HT7(b) (pKi 8.5±0.2) receptor variants and the rat native receptor (pKi 8.8±0.2). The compound displayed at least 30‐fold selectivity for the human 5‐HT7(a) receptor versus other human cloned 5‐HT receptors apart from the 5‐HT1D receptor (∼10‐fold selective).

SB‐656104‐A antagonised competitively the 5‐carboxamidotryptamine (5‐CT)‐induced accumulation of cyclic AMP in h5‐HT7(a)/HEK293 cells with a pA2 of 8.5.

Following a constant rate iv infusion to steady state in rats, SB‐656104 had a blood clearance (CLb) of 58±6 ml min−1 kg−1 and was CNS penetrant with a steady‐state brain : blood ratio of 0.9 : 1. Following i.p. administration to rats (10 mg kg−1), the compound displayed a t1/2 of 1.4 h with mean brain and blood concentrations (at 1 h after dosing) of 0.80 and 1.0 μM, respectively.

SB‐656104‐A produced a significant reversal of the 5‐CT‐induced hypothermic effect in guinea pigs, a pharmacodynamic model of 5‐HT7 receptor interaction in vivo (ED50 2 mg kg−1).

SB‐656104‐A, administered to rats at the beginning of the sleep period (CT 0), significantly increased the latency to onset of rapid eye movement (REM) sleep at 30 mg kg−1 i.p. (+93%) and reduced the total amount of REM sleep at 10 and 30 mg kg−1 i.p. with no significant effect on the latency to, or amount of, non‐REM sleep. SB‐269970‐A produced qualitatively similar effects in the same study.

In summary, SB‐656104‐A is a novel 5‐HT7 receptor antagonist which has been utilised in the present study to provide further evidence for a role for 5‐HT7 receptors in the modulation of REM sleep.

British Journal of Pharmacology (2003) 139, 705–714. doi:10.1038/sj.bjp.0705290
Effects of heparin and related molecules upon neutrophil aggregation and elastase release in vitro
Authors: Brown Rachel A , Lever Rebecca , Jones Neil A , Page Clive P
Published: June 2003

Neutrophil‐derived elastase is an enzyme implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Heparin inhibits the enzymatic activity of elastase and here we provide evidence for the first time that heparin can inhibit the release of elastase from human neutrophils.

Unfractionated and low molecular weight heparins (UH and LMWH, 0.01–1000 U ml−1) and corresponding concentrations (0.06–6000 μg ml−1) of nonanticoagulant O‐desulphated heparin (ODH), dextran sulphate (DS) and nonsulphated poly‐L‐glutamic acid (PGA) were compared for their effects on both elastase release from and aggregation of neutrophils.

UH, ODH and LMWH inhibited (P<0.05) the homotypic aggregation of neutrophils, in response to both N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP, 10−6 M) and platelet‐activating factor (PAF, 10−6 M), as well as elastase release in response to these stimuli, in the absence and presence of the priming agent tumour necrosis factor‐alpha (TNF‐α, 100 U ml−1).

DS inhibited elastase release under all the conditions of cellular activation tested (P<0.05) but had no effect on aggregation. PGA lacked efficacy in either assay, suggesting general sulphation to be important in both effects of heparin on neutrophil function and specific patterns of sulphation to be required for inhibition of aggregation.

Further investigation of the structural requirements for inhibition of elastase release confirmed the nonsulphated GAG hyaluronic acid and neutral dextran, respectively, to be without effect, whereas the IP3 receptor antagonist 2‐aminoethoxydiphenylborate (2‐APB) mimicked the effects of heparin, itself an established IP3 receptor antagonist, suggesting this to be a possible mechanism of action.

British Journal of Pharmacology (2003) 139, 845–853. doi:10.1038/sj.bjp.0705291
Calcium dobesilate potentiates endothelium‐derived hyperpolarizing factor‐mediated relaxation of human penile resistance arteries
Authors: Angulo Javier , Cuevas Pedro , Fernández Argentina , Gabancho Sonia , Videla Sebastián , Tejada Iñigo Sáenz de
Published: June 2003

We have evaluated the participation of endothelium‐derived hyperpolarizing factor (EDHF) in the endothelium‐dependent relaxation of isolated human penile resistance arteries (HPRA) and human corpus cavernosum (HCC) strips. In addition, the effect of the angioprotective agent, calcium dobesilate (DOBE), on the endothelium‐dependent relaxation of these tissues was investigated.

Combined inhibition of nitric oxide synthase (NOS) and cyclooxygenase (COX) nearly abolished the endothelium‐dependent relaxation to acetylcholine (ACh) in HCC, while 60% relaxation of HPRA was observed under these conditions. Endothelium‐dependent relaxation of HPRA resistant to NOS and COX inhibition was prevented by raising the extracellular concentration of K+ (35 mM) or by blocking Ca2+‐activated K+ channels, with apamin (APA; 100 nM) and charybdotoxin (CTX; 100 nM), suggesting the involvement of EDHF in these responses.

Endothelium‐dependent relaxation to ACh was markedly enhanced by DOBE (10 μM) in HPRA but not in HCC. The potentiating effects of DOBE on ACh‐induced responses in HPRA, remained after NOS and COX inhibition, were reduced by inhibition of cytochrome P450 oxygenase with miconazole (0.3 mM) and were abolished by high K+ or a combination of APA and CTX.

In vivo, DOBE (10 mg kg−1 i.v.) significantly potentiated the erectile responses to cavernosal nerve stimulation in male rats.

EDHF plays an important role in the endothelium‐dependent relaxation of HPRA but not in HCC. DOBE significantly improves endothelium‐dependent relaxation of HPRA mediated by EDHF and potentiates erectile responses in vivo. Thus, EDHF becomes a new therapeutic target for the treatment of erectile dysfunction (ED) and DOBE could be considered a candidate for oral therapy for ED.

British Journal of Pharmacology (2003) 139, 854–862. doi:10.1038/sj.bjp.0705293
A role of protein kinase Cμ in signalling from the human adenosine A1 receptor to the nucleus
Authors: Hill Kathryn J , Webber Anne C , Hill Stephen J
Published: June 2003

Stimulation of adenosine A1 receptors produced a stimulation of c‐fos promoter‐regulated gene transcription in Chinese hamster ovary (CHO)‐A1 cells expressing the human A1 receptor. Gene transcription was monitored using a luciferase‐based reporter gene (pGL3).

This response to the A1 receptor agonist N6‐cyclopentyladenosine (CPA) was sensitive to inhibition by pertussis toxin, the MEK‐1 inhibitor PD 98059 and by the phosphatidylinositol‐3‐kinase inhibitors wortmannin and LY 294002. The response was also completely abolished by the protein kinase C (PKC) inhibitor Ro‐31‐8220.

Several isoforms of PKC can be detected in CHO‐A1 cells (α, δ, ɛ, μ, ι, ζ), but only PKCα, PKCδ and PKC were downregulated by prolonged treatment with phorbol ester. The c‐fos‐regulated luciferase response to A1 agonists was not, however, inhibited by 24 h pretreatment with the phorbol esters phorbol 12,13‐dibutyrate (PDBu). This observation, together with the fact that a significant attenuation (40%) of the c‐fos‐luciferase response to PDBu and A1 agonist was produced by low concentrations of the PKC inhibitor Gö 6976 suggests a role for PKCμ.

Stimulation of CHO‐A1 cells with CPA stimulated the activation of endogenous PKCμ as measured by autophosphorylation. This was rapid, occurred within 1–2 min, but returned to basal levels after 30 min. Furthermore, transient expression of a constitutively active form of PKCμ resulted in a significant increase in c‐fos‐regulated gene expression.

Taken together, these data suggest that PKCμ plays an important role in the ability of the adenosine A1 receptor to signal to the nucleus.

British Journal of Pharmacology (2003) 139, 721–732. doi:10.1038/sj.bjp.0705294
Selective activation by photodynamic action of cholecystokinin receptor in the freshly isolated rat pancreatic acini
Authors: An Yu Ping , Xiao Rui , Cui Hong , Cui Zong Jie
Published: June 2003

Sulphonated aluminium phthalocyanine (SALPC) photodynamic action induces amylase secretion and permanent calcium oscillation in rat pancreatic acinar cells, because of the activation of phospholipase C or signalling proteins upstream. The aim of the present study was to investigate the involvement of muscarinic acetylcholine and cholecystokinin (CCK) receptors.

Muscarinic receptor antagonist atropine (10 μM) blocked amylase secretion induced by bethanechol (100 μM), and CCK1 receptor antagonist (S)‐N‐[1‐(2‐fluorophenyl)‐3,4,6,7‐tetrahydor‐4‐oxo‐pyrrolo‐[3,2,1‐jk][1,4] benzodiazepine‐3yl]‐1H‐indole‐2‐carboxamide (FK480) (1 μM) blocked amylase secretion induced by CCK (100 pM).

Amylase secretion induced by SALPC photodynamic action was not inhibited when atropine and FK480 were present during photodynamic action. However, addition of FK480 1 μM after initiation of photodynamic action inhibited photodynamic amylase secretion. Bethanechol (10, 100 μM) added after photodynamic action resulted in a full secretory response.

Atropine (10 nM) abolished calcium oscillation induced by bethanechol (5 μM), and FK480 (10 nM) blocked calcium oscillation induced by CCK (10 pM).

Atropine up to 10 μM was without effect on Ca2+ oscillation triggered by photodynamic action, but these oscillations were abolished by FK480 (10 nM). FK480 (10 nM) had no effect on calcium oscillations induced by bethanechol (5 μM). Bethanechol 5 μM, added after FK480 blockade of photodynamic calcium oscillation, still triggered regular calcium oscillation.

It is concluded that SALPC photodynamic action selectively and permanently activates CCK receptor in rat pancreatic acini. Such permanent and selective modulation of signalling proteins has important implications for the treatment of pancreatitis, prion diseases, and neurodegenerative disorders.

British Journal of Pharmacology (2003) 139, 872–880. doi:10.1038/sj.bjp.0705295
Role of PAF receptors during intestinal ischemia and reperfusion injury. A comparative study between PAF receptor‐deficient mice and PAF receptor antagonist treatment
Authors: Souza Danielle G , Pinho Vanessa , Soares Adriana C , Shimizu Takao , Ishii Satoshi , Teixeira Mauro M
Published: June 2003

The reperfusion of ischemic tissues may be associated with local and systemic inflammation that prevents the full benefit of blood flow restoration. The present study aimed to confirm a role for platelet‐activating factor receptor(s) (PAFR) during ischemia and reperfusion injury by using genetically modified mice deficient in the PAFR (PAFR−/− mice) and to evaluate comparatively the effectiveness of pharmacological treatment using the PAFR antagonist UK‐74,505 (modipafant).

The reperfusion of the ischemic superior mesenteric artery (SMA) induced marked local (intestine) and remote (lungs) tissue injury, as assessed by the increase in vascular permeability, neutrophil influx and intestinal hemorrhage and in the production of TNF‐α. There was also a systemic inflammatory response, as shown by the increase in serum TNF‐α concentrations and marked reperfusion‐associated lethality.

After reperfusion of the ischemic SMA, PAFR−/− mice had little tissue or systemic inflammation and lethality was delayed, but not prevented, in these mice. Interestingly, the reperfusion‐associated increases in tissue concentrations of IL‐10 were significantly greater in PAFR−/− than wild‐type mice.

Pretreatment with PAFR antagonist UK‐74,505 (1 mg kg−1) markedly prevented tissue injury, as assessed by the increase in vascular permeability, neutrophil accumulation, hemorrhage and TNF‐α concentrations in the intestine and lungs. In contrast, UK‐74,505 failed to affect reperfusion‐associated lethality and increases in serum TNF‐α when used at 1 mg kg−1.

Reperfusion‐associated lethality and increase in serum TNF‐α were only affected when a supra‐maximal dose of the antagonist was used (10 mg kg−1). At this dose, UK‐74,505 also induced a marked enhancement of reperfusion‐associated increases in tissue concentrations of IL‐10. However, at the same dose, UK‐74,505 failed to prevent reperfusion‐associated lethality in PAFR−/− mice any further.

The present studies using genetically modified animals and a receptor antagonist firmly establish a role of PAFR activation for the local, remote and systemic inflammatory injury and lethality which follows reperfusion of the ischemic SMA in mice. Moreover, it is suggested that high doses of PAFR antagonists need to be used if the real efficacy of these compounds is to be tested clinically.

British Journal of Pharmacology (2003) 139, 733–740. doi:10.1038/sj.bjp.0705296