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Background & Aims: Although nitric oxide (NO) is known to influence the recruitment of neutrophils in inflamed tissue, its role in lymphocyte-endothelial cell interactions remains poorly understood. The objectives of this study were to assess the effects of NO synthesis inhibition on T-lymphocyte migration in microvessels of rat small intestine and to define the role of adhesion molecules in this process. Methods: T lymphocytes collected from rat intestinal lymph were labeled with carboxyfluorescein diacetate succinimidyl ester and injected into the jugular vein of recipient rats. The migration of T lymphocytes into normal and N G -nitro-L-arginine methyl ester (L-NAME)-treated intestinal microvessels was monitored by using an intravital microscope. Results: L-NAME significantly increased rolling and adherence of lymphocytes in postcapillary venules of Peyer's patches and submucosal venules without significantly decreasing red blood cell velocity. The subsequent appearance of lymphocytes in the initial lymphatics was also accelerated by L-NAME. Anti–α4-integrin antibody markedly inhibited the L-NAME–induced lymphocyte-endothelial cell interaction. Anti–P-selectin monoclonal antibody also significantly attenuated these adhesive interactions in both vascular regions. Conclusions: These data suggest that NO is an important modulator of lymphocyte migration in Peyer's patches and in nonlymphoid regions of the intestine. GASTROENTEROLOGY 1998;115:618-627

BACKGROUND & AIMS: Although dietary fat is known to modulate immune functions, there is a paucity of data concerning the effects of fat absorption on migration of lymphocytes in intestinal lymphoid tissue. The objective of this study was to assess the influence of fat absorption on T-lymphocyte migration in Peyer's patches. METHODS: T lymphocytes collected from rat intestinal lymph were labeled with carboxyfluorescein diacetate succinimidyl ester and injected into the jugular vein of recipient rats. Olive oil or octanoic acid was placed into the duodenum, and Peyer's patch microcirculation was observed by intravital fluorescence microscopy. RESULTS: Rolling and adherence of lymphocytes in postcapillary venules were noted in both fasted and fed rats. However, lymphocyte adherence was significantly greater in olive oil-fed rats than in fasted rats. Olive oil also significantly increased the transendothelial migration of T lymphocytes. Octanoic acid stimulated lymphocyte rolling but did not affect lymphocyte adherence. Olive oil enhanced the expression of both alpha4-integrin and L-selectin on lymphocytes, whereas octanoic acid only increased the expression of L-selectin. CONCLUSIONS: Lymphocyte rolling and adherence are enhanced by olive oil feeding, possibly through an interaction between activated adhesion molecules on lymphocytes and counter- receptors on endothelial cells. (Gastroenterology 1997 Mar;112(3):813-25)

Background & Aims: The basic mechanisms of food allergies are still unknown. The aims of this study were to investigate whether endothelins (ETs) in the intestinal mucosa are involved in the pathogenesis of intestinal anaphylaxis. Methods: Sprague–Dawley rats were sensitized to chicken egg albumin (EA) by intraperitoneal injection. Fourteen days after sensitization, EA was administered in the jejunal segments to induce intestinal anaphylaxis. Net water outflux and histamine release into loops and serum concentrations of rat mast cell protease II (RMCP-II) were determined. ET-1 and ET-3 concentrations in the jejunal mucosa were determined, and expression of the corresponding messenger RNAs was examined by competitive polymerase chain reaction. Results: In sensitized animals, challenge with intraluminal antigen caused a significant increase in net water outflux and histamine release together with an elevation of serum RMCP-II concentrations. Mucosal concentrations of ET-1 and ET-3 and expression of their messenger RNAs were significantly increased in sensitized animals after EA challenge. Treatment with an ET A -receptor antagonist, but not an ET B -receptor antagonist, attenuated the increase in net water outflux, histamine release, and serum RMCP-II concentrations in rats with EA-induced intestinal anaphylaxis. Conclusions: Release of ETs in the intestinal mucosa increased in sensitized animals after EA challenge. ETs may play a significant role in the development of intestinal anaphylaxis via an ET A receptor. GASTROENTEROLOGY 1998;115:348-356

Background & Aims : Although recirculation of lymphocytes through Peyer's patches is important for specific immune defense, the intraorgan migration of lymphocyte subpopulations has not been clearly understood. The aim of this study was to compare the spatial distributions of labeled lymphocytes among various subpopulations in rat Peyer's patches. Methods : Lymphocytes collected from intestinal lymph were separated into CD4+, CD8+, and T and B cells, labeled with a fluorochrome carboxyfluorescein diacetate succinimidyl ester, and injected into the jugular vein. Peyer's patches of recipient rats were observed by intravital fluorescence microscopy. Results : No significant difference was found in the percentage of lymphocytes in transit or in the rolling velocity among different subpopulations. Lymphocytes sticking to the venules increased in number at 10–20 minutes, with preferential adherence of CD4+ cells to venules of 25–50 μm and preferential adherence of B cells to the venules of a wider size range. After 30 minutes, extravasated lymphocytes moved into the interstitium. B cells migrated from venules more quickly than CD4+ cells. CD8+ cells showed an intermediate pattern between CD4+ and B cells in sticking and migratory behaviors. Subsequently, CD4+ and CD8 cells preferentially appeared in parafollicular microlymphatics. Conclusions : Significant differences were observed among lymphocyte subpopulations in terms of spatial distribution of lymphocytes sticking to venules, migration into the interstitium, and their lymphatic transport.

BACKGROUND: Genus Lactobacillus is classified broadly into two categories. One is isolated from dairy products or gut and another is vegetable -derived Lactobacillus (VDL) isolated from fermented vegetable food such as pickles or a soy-sauce. Genus Lactobacillus isolated from dairy products or gut have been used for clinical trials in inflammatory bowel disease (IBD) as probiotics, however, VDL was poorly investigated. In this study, we newly isolated two VDL from Funazushi (Japanese old-style sushi; salted and fermented with a rice and a crusian carp) and examined its anti-inflammatory effect on dextran sodium sulfate (DSS)induced colitis. MATERIALS & METHODS: The surrounding rice material of Funazushi, was homogenized and plated on MRS medium for selection of Genus Lactobacillus. A single colony was picked and sorted out by its ability for suppression of proinflammatory cytokines at mRNA level in RAW264.7 cells after LPS stimulation (In Vitro screening). Intragastric administration of Lactobacillus isolated from Funazushi or L.gasseri (ATCC 33323) was performed in 8-week olds-female C57BL/6 mice every day during administration of 6% DSS in water for 7 days. The degree of colonic inflammation and mRNA levels of expression of cytokines in colonic mucosa were determined. Results: Among 500 screened colonies, two different anti-inflammatory types of Lactobacillus were isolated from Funazushi (s193 and s292). Thus, in addition to each Lactobacillus alone administration, we performed the combinatory administration with equivalent amount of s193 and s292 (dual administration). Although each Lactobacillus from Funazushi showed an attenuating effect on inflammation, dual administration significantly improved survival rate, body weight loss and histologic damage score in mice compared with either DSS-treated control, L.gasseri, s193 or s292 alone group. mRNA level of IL-6 was attenuated by s193 alone, and IL-12p40 or IL-23p19 level was decreased by s292 alone. mRNA levels of IL-1β and TNF-α were also remarkably inhibited by dual administration. Conclusions: Two strains of newly established Lactobacillus isolated from Funazushi showed a different anti-inflamattory effect on DSS-induced colitis and that effect was enhanced by simultaneous dual administration. These data suggested that VDL isolated from Funazushi may be useful for treatment for IBD with stronger antiinflammatory effects than Lactobacillus isolated from dairy products or gut.

[Backgroundl Most cases of pancreatic cancer are inoperable when diagnosed. In these cases immunotherapy, which targets cancer cells, is one of the potent options. On the other hand, anti-anglogenic therapy, which targets tumor vessels, has been reported to be promising for solid tumors. However, single therapy of these has not induced tumor regression within a therapeutic dose. Therefore, in this study, we examined whether the combination of immunotherapy with DCs and anti-angiogenetic drug TNP-470 induces tumor regression and tested the effectiveness of tumor lysate (TL) pulsation for DCs. [Matenals& Methods] 3x105 mouse pancreatic adenocarcinoma cells (pan02 origanated rum C57/BL6) were orthotopically inoculated into the pancreas of C57/BL6 mouse 3~4 weeks of age. Tumor take ratio was 100% at 6 weeks. 3xl04DCs were injected into peritoneal cavity at 4 and 5weeks. TNP-470 (100mg/kg/week) was injected subcutaneously into tumor-bearing mice every other day from 4 weeks to 6weeks. We measured tumor volume, vascular density, and vessel diameter by the injection of FITC-dextran using intravital microscope at 6weeks. Proliferation and apoptosis of cancer cells were evaluated by PCNA and TUNEL. We compared anticancer effect among 5 groups, which are DCs with TL (+TL), DCs without TL (-TL), DCs with TL +TNP-470 (TNP+TL), DCs without TL + TNP-470 (TNP-TL), and control (without DC and without TNP-470). [Results] Tumor volume was 22.5 +_ 7.6ram 3 in + TL, 2 6 7 _ + i1 0mmqn -TL, 4.8_+2.5 mm ~ in TNP+ TL, and 31.2 _+ 20.4mm 3 in TNPTL, and these numbers were significantly smaller compared with 113.7 _+ 38.6mm3in control. Tumor vascular density was 36.4 _+ 4.4cm/cm z in + TL, 33.8-+ 5.6cm/cm 2 in -TL, 21.3 _+ 4.3cm/cm: in TNP + TL, and 28.5 _+ 4.4cm/cm z in TNP-TL, and these numbers were significantly smaller than 88.8-+ 7.3cm/cm 2 in control. Tumor vessel diameter was 9.9-+ 19/*m in TNP+TL, 8.5 +0 .8~m in TNP-TL and these data were smaller than 22.1 _+ 1.0~min control, J.8.9_+ 2.9~m in + TL, and 17.3 _+ 3.7/*m in -TL. There were not significant differences in proliferation among 5 groups, however, the number of apoptotic cells tend to be larger in +TL groups than -TL groups. There were not oh',nons side effects in all groups. [Conclusion] Data suggested that tumor-lysate pulsed DCs combined with FNP-470 induced regression of orthotopic pancreatic cancer growth, possibly through the induction of cancer cell apoptosis and anti-angiogenic effect.

Although recirculation of lymphocytes through intestinal mucosa is important for specific immune defense, homing of lamina proprial lymphocytes (LPLs) has not been clearly understood. The aim of this study was to compare the dynamic process of lymphocyte-endothelium recognition and binding in murine intestinal mucosa between lymphocytes from lamina propria of intestine and spleen under intravital microscope. Methods: BALB/c mice were used in this study. LPLs were separated from non-lymphoid area of small intestine by collagenase treatment and Percoll centrifugation. Ileal Peyer's patches, villus mucosa and spleen were observed under an intravital fluorescence microscope. Lymphocytes collected from lamina propria and spleen were fluorescence labeled and injected into the jugular vein of recipient mice. The cell kinetics of infused lymphocytes were monitored in these organs at 10min-intervals for 60 min. Surface expression of adhesion molecules (ct4-, and 137-integrin, CD 1 la and L-selectin) on lymphocytes was determined by FACScan. Results: By observation of villus mucosa LPLs were accumulated in the microvessels of villus tip and the number was gradually increased time-dependently up to 60 min. These accumulation was significantly inhibited by the treatment of lymphocytes with anti-[37. A small number of lymphocytes were attached to capillaries of crypt base, but there was no adherence of LPLs to submucosal venules of the intestinal mucosa or postcapillary venules of Peyer's patches during the entire course of observation. On the other hands, splenic lymphocytes was shown to adhere selectively to the postcapillary venules of Peyer's patches. In the villus mucosa, some splenic lymphocytes were adhered to microvessels of villus tip and crypt base for the first 10 min, but these lymphocytes were often detached and the number of adherent lymphocytes was decreased at 60 min. LPLs were also found in splenic microvessels, but the number was smaller than those of splenic lymphocytes. By FACscan analysis, decreased expression of L-selectin and increase in 137 expression were observed in LPLs as compared with splenic lymphocytes while CD11a and et4-integrin were not changed. Conclusions: It was demonstrated in vivo for the first time that lymphocytes from lamina proptia adhered selectively to the microvessels of villus tip O f intestine possibly via 137, but not to the postcapillary venules of Peyer's patches in contrast to splenic lymphocytes.