Role of the gut-associated and secondary lymphoid tissue in the induction of chronic colitis.

BACKGROUND: It is well known that enteric bacterial antigens drive the development of chronic colitis in a variety of different mouse models of the inflammatory bowel diseases (IBD). The objective of this study was to evaluate the role of gut-associated lymphoid tissue (GALT; Peyer's patches, isolated lymphoid follicles), mesenteric lymph nodes (MLNs) and spleen in the pathogenesis of chronic colitis in mice. METHODS: Surgical as well as genetic approaches were used to generate lymphopenic mice devoid of one or more of these lymphoid tissues. For the first series of studies, we subjected recombinase activating gene-1-deficient mice (RAG(-/-) ) to sham surgery (Sham), mesenteric lymphadenectomy (MLNx), splenectomy (Splx) or both (MLNx/Splx). In a second series of studies we intercrossed lymphotoxinß-deficient (LTß(-/-) ) mice with RAG(-/-) animals to generate LTß(-/-) x RAG(-/-) offspring that were anticipated to contain functional MLNs but be devoid of GALT and most peripheral lymph nodes. Flow purified naïve (CD4(+) CD45RB(high) ) T-cells were adoptively transferred into the different groups of RAG(-/-) recipients to induce chronic colitis. RESULTS: We found that at 3-5 wks following T-cell transfer, all four of the surgically-manipulated RAG(-/-) groups (Sham, MLNx, Splx and MLNx/Splx) developed chronic colitis that was similar in onset and severity. Flow cytometric analysis revealed no differences among the different groups with respect to surface expression of different gut-homing markers nor were there any differences noted in IFN-γ and IL-17 generation by mononuclear cells isolated among these surgically-manipulated mice. Although we anticipated that LTß(-/-) x RAG(-/-) mice would contain functional MLNs but be devoid of GALT and peripheral lymph nodes (PLNs), we found that LTß(-/-) x RAG(-/-) mice were in fact devoid of MLNs as well as GALT and PLNs. Adoptive transfer of CD45RB(high) T-cells into LTß(-/-) x RAG(-/-) mice or their littermate controls (LTß(+/+) x RAG(-/-) ) induced rapid and severe colitis in both groups. CONCLUSIONS: Taken together, our data demonstrate that: a) neither the GALT, MLNs nor PLNs are required for induction of chronic gut inflammation in this model of IBD and b) T-and/or B-cells may be required for the development of MLNs in LTß(-/-) mice.

Lemon grass (Cymbopogon citratus) ameliorates murine spontaneous ileitis by decreasing lymphocyte recruitment to the inflamed intestine.

OBJECTIVE: Aberrant leukocyte migration has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Lemon grass is a natural herb that contains citral, which suppresses lymphocyte expression of gut homing molecules by inhibiting retinoic acid formation. We therefore hypothesized that lemon grass intake could ameliorate excess migration of leukocytes to the inflamed intestine in chronic ileitis. METHODS: Migration of fluorescence-labeled T cells to microvessels in the ileal mucosa of SAMP1/Yit mice was monitored using intravital microscopy. In some mice, lemon grass solution was administered for two weeks. For evaluation of the effects on chronic ileitis, mice were treated with lemon grass for 26 weeks. RESULTS: Surface expression of beta7 and CCR9 on T lymphocytes was stronger in SAMP1/Yit mice than in AKR/J mice. Lemon grass treatment attenuated the surface expression of beta7-integrin and CCR9. The number of adherent lymphocytes to microvessels in chronic inflamed ileum was significantly few when lymphocytes were isolated from lemon grass treated mice. Long-term lemon grass treatment improved ileitis in SAMP1/Yit mice, which was assessed by body weight, histological changes and the infiltration of beta7-positive cells. CONCLUSION: Lemon grass ameliorated ileitis through decreasing lymphocyte migration by inhibiting beta7-expression, suggesting its therapeutic usefulness for IBD.

Mutational analysis of the PTEN gene in women with premature ovarian failure.

Recent studies in mammals have suggested that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) - phosphatidylinositol-3, 4, 5-trisphosphate pathway in oocytes might be related to the pathogenesis of premature ovarian failure (POF). The aim of this study was to investigate whether mutations of the PTEN gene are present in women with POF. We analyzed the coding region of the PTEN gene in 20 women with idiopathic POF and 20 normal controls. The PTEN gene was amplified by the polymerase chain reaction using genomic DNA isolated from blood samples. Amplified DNA was analyzed by denaturing gradient gel electrophoresis and direct sequencing. No causative mutation was detected in the coding regions of this gene. Although we found a point variation in exon 7 of one POF patient, this was a single nucleotide polymorphism that has already been reported.

Oral tolerance induced by enterobacteria altered the process of lymphocyte recruitment to intestinal microvessels: roles of endothelial cell adhesion molecules, TGF-beta and negative regulators of TLR signaling.

OBJECTIVE: Although enterobacteria are implicated in intestinal immune response, there has been no report on how intraluminal pathogens affect lymphocyte recruitment. The aim of this study was to determine how the presence of intestinal flora affects lymphocyte migration to intestine under physiological and lipopolysaccharide (LPS)-induced inflammatory conditions. METHODS: Interaction of T-cells with ileal microvessels was monitored by using an intravital microscope in mice under germ-free (GF) and specific pathogen-free (SPF) conditions. LPS was administered into either the peritoneal cavity or duodenum before lymphocyte injection. RESULTS: Adherence of T-cells was greater in SPF than in GF mice, indicating that the presence of enterobacteria upregulated migration under physiological conditions. Intraperitoneally administered LPS significantly increased the adherence of T-cells in both GF and SPF mice accompanied by the expression of adhesion molecules and proinflammatory cytokines. However, intraluminally administered LPS did not enhance the adherence of T-cells in SPF mice. A significant induction of increase in mRNA expression of IRAK-M, a negative regulator of TLR4 signaling, and transforming growth factor beta (TGF-beta), a regulatory cytokine, was observed in SPF mice after luminal LPS treatment. CONCLUSIONS: Tolerance to intraluminally administered LPS in the lymphocyte recruitment process was induced by enterobacteria, possibly via the induction of IRAK-M and TGF-beta.

Long-term outcome of endoscopic semiconductive diode laser irradiation therapy with injection of indocyanine green for early gastric cancer.

BACKGROUND AND AIM: Semiconductive laser irradiation has been used to treat early gastric cancer. However, the long-term follow up results have not been reported. The objective of the present study was to assess retrospectively the clinical usefulness of diode laser irradiation for early gastric cancer. METHODS: The subjects of this study were 13 patients (14 lesions) selected from 125 patients with early gastric cancer who were treated by endoscopy during the period from September 1995 to February 2003. The macroscopic tumor type was superficial type, including eight lesions of 0'-IIc and six lesions of 0'-IIa. Histological diagnoses were eight cases (nine lesions) of well-differentiated adenocarcinoma, three cases of moderately differentiated adenocarcinoma and two cases of poorly differentiated adenocarcinoma. After injection with indocyanine green solution (1 mg/mL) into the submucosal layer, a semiconductive diode laser (30-40 W/s) was irradiated by the non-contacting method. RESULTS: The total amount of laser irradiation for 14 lesions was 9568.80 +/- 7197.01 J on average. There was no major complication. In the period up to December 2007, six patients survived and seven patients died. However, no-one died of progression of gastric cancer. The mean survival times of all patients, survivors and patients who died were 5 years 2.8 months; 6 years 4.5 months; and 4 years 11.7 months, respectively. CONCLUSIONS: Early gastric cancer can be successfully treated by laser therapy with few complications and good prognosis. This method is expected to be most suitable and effective for elderly patients with serious underlying disease.

P-selectin-dependent monocyte recruitment through platelet interaction in intestinal microvessels of LPS-treated mice.

BACKGROUND: Although platelets or monocytes are thought to be involved in intestinal inflammation, there has been no report on whether platelets can modulate monocyte recruitment in intestinal microvessels. The objective of this study was to determine whether blockade of platelet adhesion attenuates monocyte recruitment in inflamed murine intestinal microvessels. METHODS: Monocytes and platelet-rich plasma were obtained from C57B6/J mice. Interaction of monocytes and platelets with intestinal microvessels was observed under an intravital microscope. Lipopolysaccharide (LPS) was administered intraperitoneally. The effects of anti-P-selectin or anti-platelets antibody treatments or phosphodiesterase (PDE) inhibitors (PDE-3 and PDE-2/4 inhibitor) treatments were also studied. RESULTS: LPS-treatment increased the rolling and adhesion of both platelets and monocytes. Pretreatment with an anti-P-selectin antibody inhibited the increased platelet adhesion to venular walls and also attenuated the monocyte adhesion. A PDE-2/4 inhibitor (ibuzilast) also ameliorated both platelet and monocyte adhesion. A PDE-3 inhibitor (cilostazol) ameliorated only monocyte adhesion without directly affecting the adhesion of platelets to microvessels. CONCLUSIONS: We observed inhibition of platelets adhesion attenuated monocytes recruitment in intestinal microvessels. Attenuation of LPS induced monocyte adhesion by a specific PDE-3 inhibitor suggests that P-selectin on activated platelets may play an important role through monocyte and platelet interaction.

Omega-3 fatty acids exacerbate DSS-induced colitis through decreased adiponectin in colonic subepithelial myofibroblasts.

BACKGROUND: Although the immunoregulatory effects of omega-3 fatty acid and adiponectin have been postulated, their role in intestinal inflammation is controversial. The aim of this study was to determine whether dietary fat intake influences activity of colonic inflammation through modulating this system. METHODS: C57BL/6 mice received dextran sulfate sodium for induction of colitis. Mice were fed a control diet, omega-3 fat-rich diet, omega-6 fat-rich diet, or saturated fat-rich diet. Some mice were administered a peroxisome proliferator activated receptor-gamma; agonist, pioglitazone. Messenger RNA expression of adiponectin and its receptors were analyzed. Adiponectin expression in colonic mucosa of ulcerative colitis patients was also analyzed. RESULTS: The receptors for adiponectin were found to be ubiquitously expressed in epithelial cells, intraepithelial lymphocytes, lamina proprial mononuclear cells, and subepithelial myofibroblasts from colonic tissue, but adiponectin was only expressed in myofibroblasts. Induction of colitis significantly decreased the expression of adiponectin in colonic mucosa. The omega-3 fat diet group, but not the other fat diet groups, showed exacerbated colitis with a further decrease of adiponectin expression. Pioglitazone treatment ameliorated the level of decrease in adiponectin expression and improved colonic inflammation induced by the omega-3 fat-rich diet. In patients with ulcerative colitis, the expression level of adiponectin in colonic mucosa was also decreased compared with that in control mucosa. CONCLUSIONS: Adiponectin was found to be expressed in myofibroblasts. Adiponectin expression was significantly suppressed by induction of colitis, and aggravation of colitis after exposure to omega-3 fat may be due to a further decrease in the expression level of adiponectin.

Concomitant treatment of severe uterine adenomyosis in a premenopausal woman with an aromatase inhibitor and a gonadotropin-releasing hormone agonist.

OBJECTIVE: To assess the effect of aromatase inhibitors with GnRH agonist for a severe symptomatic adenomyosis that is refractory to GnRH agonist and danazol with GnRH agonist. DESIGN: Case report. SETTING: Clinical practice in university hospital. PATIENT(S): A 34-year-old woman with a complaint of severe dysmenorrheal, symptomatic anemia, and a desire to retain fertility. INTERVENTION(S): Aromatase inhibitor anastrozole given orally (1.0 mg or 2.0 mg daily) for 16 weeks and GnRH agonist given monthly (injected SC, 1.8 mg) for 4 months. MAIN OUTCOME MEASURE(S): Measurements of uterine volume and levels of serum E(2), estrone, A, dehydroepiandrosterone sulfate, LH, FSH, and CA125. RESULT(S): Uterine volume was reduced. The reduction rate of uterine volume estimated by magnetic resonance imaging and ultrasonography was 60% after 8 weeks of treatment. CONCLUSION(S): Aromatase inhibitor with GnRH agonist therapy was useful for the management of a severely adenomyotic woman whose desire was for conservative treatment.

Effectiveness of antiadhesion barriers in preventing adhesion after myomectomy in patients with uterine leiomyoma.

BACKGROUND: Myomectomy often causes adhesion formation and decreases subsequent fertility. The purpose of the present study was to evaluate the effectiveness of several antiadhesion barrier materials in preventing adhesion after myomectomy. METHODS: We prospectively classified 63 women undergoing myomectomy alone into four groups according to the type of antiadhesion material used: Hyaluronic acid-carboxymethylcellulose film (Seprafilm) (n = 21, Group 1), Dextran 40 (10% Dextran 40 Low Injection) (n = 17, Group 2), factor 13 with fibrinogen (Beriplast) (n = 12, Group 3) and control (n = 13, Group 4). We performed early second-look laparoscopy after the seventh post-operative day in all patients and examined adhesion formation in the abdominal cavity. The incidence of adnexal adhesions was evaluated according to the American Fertility Association (AFS) adhesion score. RESULTS: The incidence of uterine adhesion was 14.3% in Group 1, 70.6% in Group 2, 75.0% in Group 3 and 76.9% in Group 4. Adhesion formation in Group 1 was significantly less than that in Group 2 (p = 0.0004), Group 3 (p = 0.0005) and Group 4 (p = 0.0003). The incidence of peritoneal adhesion was 14.3% in Group 1, 29.4% in Group 2, 41.6% in Group 3 and 69.2% in Group 4. Adhesion formation in Group 1 was significantly less than that in Group 4 (p = 0.001). AFS scores in Groups 1-4 were 0.38+/-1.02, 4.58 +/- 7.02, 0.83 +/- 1.99 and 8.53 +/- 8.79 (mean +/- S.D.), respectively. Group 1 had the lowest AFS score and the difference between Group 1 and Group 4 was significant (p < 0.0001). The AFS score in Group 3 was also significantly less than that of Group 4 (p = 0.0009). CONCLUSION: Seprafilm was highly effective and was superior to the other antiadhesion materials tested in preventing uterine adhesions after myomectomy.

Interaction of presenilins with FKBP38 promotes apoptosis by reducing mitochondrial Bcl-2.

Presenilins 1 and 2 (PS1/2), causative molecules for familial Alzheimer's disease (FAD), are multipass transmembrane proteins localized predominantly in the endoplasmic reticulum (ER) and Golgi apparatus. Heteromeric protein complexes containing PS1/2 are thought to participate in several functions, including intramembrane proteolysis mediated by their gamma-secretase activities. Previous studies have shown that PS1/2 are also involved in the regulation of apoptotic cell death, although the underlying mechanism remains unknown. Here, we demonstrate that FKBP38, an immunophilin family member residing in the mitochondrial membrane, is an authentic PS1/2-interacting protein. PS1/2 and FKBP38 form macromolecular complexes together with anti-apoptotic Bcl-2. PS1/2 promote the degradation of FKBP38 and Bcl-2 and sequester these proteins in the ER/Golgi compartments, thereby inhibiting FKBP38-mediated mitochondrial targeting of Bcl-2 via a gamma-secretase-independent mechanism. Thus, PS1/2 increase the susceptibility to apoptosis by antagonizing the anti-apoptotic function of FKBP38. In contrast, C-terminal fragments of caspase-processed PS1/2 redistribute Bcl-2 to the mitochondria by abrogating the activity of full-length PS1/2, resulting in a dominant-negative anti-apoptotic effect. In cultured cells and mutant PS1-knockin mice brains, FAD-linked PS1/2 mutants enhance the pro-apoptotic activity by causing a more efficient reduction in mitochondrial Bcl-2 than wild-type PS1/2. These results suggest a novel molecular mechanism for the regulation of mitochondria-mediated apoptosis by competition between PS1/2 and FKBP38 for subcellular targeting of Bcl-2. Excessive pro-apoptotic activity of PS1/2 may play a role in the pathogenesis of FAD.

Fetal hemolytic disease due to anti-Rh17 alloimmunization.

OBJECTIVE: To delineate clinical features of a case of fetal hemolytic disease due to anti-Rh17, along with a review of relevant studies published in English and Japanese. METHODS: We present clinical features of a -D-/-D- phenotype woman with anti-Rh17 alloimmunization during pregnancy. Relevant English literature in the MEDLINE database was reviewed, while Japanese studies were searched in the Japana Centra Revuo Medicina database. RESULTS: A Japanese -D-/-D- woman with anti-Rh17 (Hro) was treated during pregnancy. Serial ultrasonography, antibody titers, amniocenteses, and cordocenteses were conducted for perinatal management. Amniocentesis results demonstrated a high delta optical density level of 450 in the amniotic fluid, while cordocentesis revealed alloimmunization between the mother and the fetus as well as fetal hemolytic anemia. Blood flow velocity in the middle cerebral artery indicated a rapid development of fetal anemia. The newborn demonstrated severe anemia and hyperbilirubinemia, which were successfully treated with exchange transfusions. Two cases of prenatally diagnosed fetal hemolytic disease due to anti-Rh17 were found published in English and 5 in Japanese. CONCLUSION: A -D-/-D- phenotype patient with anti-Rh17 was successfully managed during pregnancy and a good outcome for the neonate was achieved. Our results and a review of related literature led to the following suggestions. The first pregnancy in a -D-/-D- woman may be affected, an anamnestic immune response can easily occur during pregnancy, the level of anti-Rh17 titer is indicative of the degree of fetal hemolysis, and appropriate intrauterine intervention is warranted for achievement of a good outcome.

Follistatin-related gene (FLRG) expression in human endometrium: sex steroid hormones regulate the expression of FLRG in cultured human endometrial stromal cells.

Follistatin-related gene (FLRG) encodes a novel secreted glycoprotein that is highly homologous to follistatin and binds activins and bone morphogenetic proteins, members of the TGF beta superfamily of growth/differentiation factors. FLRG protein inhibits activin-induced and bone morphogenetic protein-2-induced transcriptional responses in a dose-dependent manner, and its mRNA is abundantly expressed in human placenta, heart, aorta, testis, and adrenal gland. In this study we showed that FLRG mRNA was expressed in human endometrium across the menstrual cycle and in decidua of early pregnancy. In the proliferative phase of the menstrual cycle, FLRG protein was detected predominantly in the cytoplasm of endometrial epithelium. In the secretory phase and in early pregnancy, it was also detected in the nuclei of endometrial stromal cells. Using in vitro decidualization model, we demonstrated that 17 beta-estradiol plus progesterone, but not 17 beta-estradiol or progesterone alone, induced FLRG expression significantly. These results suggest that FLRG expression in endometrial stromal cells is regulated by the concerted action of ovarian steroid hormones via decidualization, and FLRG protein may participate in the regulation of stromal cell decidualization as a binding protein for members of TGF beta superfamily.

Effectiveness of 2-step (consecutive) embryo transfer. Comparison with cleavage-stage transfer.

OBJECTIVE: To evaluate the effectiveness of 2-step (consecutive) embryo transfer (ET), in which two cleaved embryos are transferred on day 2 (first step of ET) and a single blastocyst is transferred on day 5 (second step of ET) in comparison with conventional day 2 ET. STUDY DESIGN: Observational comparative study at different time periods. Subjects were those who had > or = 4 embryos on day 2 after oocyte retrieval. Those in the conventional group (n = 60) received conventional day 2 ET, in which 3 cleaved embryos were transferred on day 2 between January 1999 and June 2000. Those in the 2-step group (n = 124) received 2-step embryo transfer, in which 2 cleaved embryos were transferred on day 2 and a blastocyst was transferred on day 5 between September 2000 and February 2002. Patients who planned to receive 2-step ET but could not proceed to the second step of ET because of failure to produce blastocysts were included in the 2-step group. RESULTS: The ongoing pregnancy rate with 2-step ET (51.6%) was significantly higher than with conventional ET (26.7%) (P < .01). The clinical pregnancy rate and implantation rate in conventional and 2-step ET were 30.0% vs. 59.7% and 11.1% vs. 27.8%, respectively (P < .001). CONCLUSION: Two-step ET may benefit patients who can obtain a sufficient number of embryos.

Protein S gene mutation in a young woman with type III protein S deficiency and venous thrombosis during pregnancy.

BACKGROUND: We attempted to identify a gene defect in a young woman with type III protein S deficiency and venous thrombosis during pregnancy. METHODS: Measurements of total and free PS antigen levels in plasma were carried out using an enzyme-linked immunosorbent assay. Plasma PS cofactor activity was determined by a clotting assay using activated factor V as the substrate. Genomic DNA prepared from peripheral blood was amplified by polymerase chain reaction (PCR) with PROS1-specific oligonucleotide primers. PCR products were sequenced on both strands using specific oligonucleotide primers. RESULTS: Plasma PS cofactor activity was undetectable in every measurement at 36 weeks of gestation, as well as at 2 weeks and 4 months after delivery. Plasma total PS antigen levels were 70% and 67% at 2 weeks and 4 months after delivery, respectively. Free PS antigen level was 24% at 4 months after delivery. Of all exons analyzed, codon 295 of GGC in exon 10 was substituted for AGC. This missense mutation predicted an amino acid change of glycine to serine. CONCLUSIONS: Measurements of total and free PS antigen levels along with PS activity indicated that this was a case of type III PS deficiency. DNA analysis identified a heterozygous missense mutation of codon 295 in the PS gene, substituting glycine for serine.