FOXP3 expression in duodenal mucosa in pediatric patients with celiac disease.

OBJECTIVE: It was the aim of this study to evaluate the number of 2 lymphoid subpopulations, CD8(+) cells and FOXP3(+), in the duodenum mucosa from pediatric celiac patients. METHODS: Tissue sections prepared from paraffin-embedded biopsies of the descending duodenum of 61 celiac patients with Marsh grade 1 (M1), M2 and M3 disease and biopsies from 21 age-matched non-celiac (NC) patients were immunohistostained with anti-CD8 or FOXP3 antibodies. RESULTS: The histological Marsh grade correlated with the mean number of FOXP3(+) cells in the lamina propria (LP) mucosa (8.9 ± 1.1, 6.8 ± 2.4, 24.5 ± 2.6 and 31.1 ± 2.8 for NC, M1, M2 and M3 biopsies, respectively; p < 0.001). Using a cutoff point of 15 cells, 95% of NC and 88% of M1 biopsies had a mean of <15 FOXP3(+) cells compared with 14% for M2 and 13% for M3 biopsies. The number of FOXP3(+) cells in the epithelial mucosa also correlated with transglutaminase type 2 serum levels from the celiac patients. Unlike the FOXP3(+) cells, CD8(+) lymphocytes were present in both LP and surface epithelial mucosa and significantly different only in the LP mucosa of the M2 and M3 groups. CONCLUSION: The number of FOXP3(+) cells is substantially increased in the mucosa of celiac patients at advanced stages. Characterization of the activity of these cells in celiac and in other inflammatory bowel diseases will enable us to understand the significance of these cells in celiac disease.

Effects of 5-FU on DNA synthesis and cytotoxicity of human lymphocytes induced by IL-2, TGF-beta3 and PGE2.

BACKGROUND: Low 5-fluorouracil (5-FU) concentrations cause a significant increase in DNA synthesis in mitogen-activated human lymphocytes. MATERIALS AND METHODS: We explored 2.5 microM 5-FU-induced DNA synthesis by testing 5-FU activity in hypoxanthine-aminopterin-thymidine (HAT)-containing medium, and its effect on thymidylate synthase (TS) activity and CD25 expression in interleukin (IL)-2-activated human peripheral blood mononuclear cells (PBMCs) and the combined effects with prostaglandin E(2) (PGE(2)) and transforming growth factor (TGF)-beta3. RESULTS: The co-stimulatory effect of 2.5 microM 5-FU on DNA synthesis was abrogated in HAT-cultured medium. 5-FU substantially reduced TS activity by 50% in IL-2-activated PBMCs. 5-FU combined with TGF-beta3 and PGE(2) did not alter their inhibitory effects on IL-2-activated natural killer cell cytotoxicity, but substantially affected increased DNA synthesis of cells cultured in IL-2 and co-cultured with 10 ng/ml TGF-beta3 and 10 microM PGE(2). CONCLUSION: Low 5-FU concentrations increase DNA synthesis in lymphocytes and exert a co-stimulatory activity on TGF-beta3 and PGE(2) modulation of IL-2-activated lymphocytes.

Galectin-3 expression in pouchitis in patients with ulcerative colitis who underwent ileal pouch-anal anastomosis (IPAA).

Galectin-3, an endogenous pleiotropic beta-galactoside-binding protein, which is expressed by various malignant and normal cells, regulates many biological and pathological processes, including inflammation. In the present study, we tested a possible correlation between the severity of pouchitis in patients with ulcerative colitis who underwent ileal pouch-anal anastomosis (IPAA) and the presence of galectin-3(+) macrophages in pouch mucosa. Paraffin-embedded pouch biopsies from patients with normal pouch function or chronic and recurrent acute pouchitis were immunohistostained with galectin-3, CD68, and smooth muscle actin (SMA) antibodies. Microscopic examination was performed in a blinded fashion. There was a significant decrease in the staining index of galectin-3 in the subepithelial macrophages in patients with chronic pouchitis (0.53, P=0.001; n=12) or recurrent acute pouchitis (0.43, P=0.008; n=10) when compared to patients with no clinical manifestations of pouchitis (0.63, n=12). No significant differences were noted in the lamina propria of small intestine biopsies from the same patients (from 0.63 to 0.68, P=0.24). Galectin-3 staining was restricted to CD68(+) macrophages and not present in myofibroblasts. Clinical manifestation of pouchitis is inversely correlated with galectin-3 expression in the pouches' subepithelial lamina propria macrophages.

A novel assessment of the quality of immunohistostaining overcomes the limitations of current methods.

Quality assurance has become an integral part of surgical pathology. Despite the development of interdisciplinary quality systems, however, the means for objective analysis in surgical pathology are limited. Immunohistostaining is a multi-factorial procedure that depends on the quality of reagents and antibodies employed in the process and on technical methodology. In the present study, we aim to establish a straightforward procedure for objective quality evaluation of the components involved in immunohistostaining. The quality of two of these components, the primary antibody and the automated staining device, was assessed by employing each component from two different sources, one serving as the test substance and the second as the reference. Assessment was performed by at least two pathologists in a blinded fashion using pre-established quality criteria and scores. The quality analysis of two automated devices revealed a significant difference between the reference and tested devices (3.5+/-1.7 and 4.2+/-1.5, respectively, P>0.05), while the analysis of two selected antibodies did not reveal any statistical difference. The described method provided objective quality assessment of selected components affecting immunohistostaining by elaborating numeric values that enabled statistical analysis. This approach is applicable to any given component in various surgical pathology procedures.

Effects of 5-fluorouracil on human mitogen-activated peripheral blood lymphocytes from healthy individuals.

BACKGROUND: The effect of 5-fluorouracil (5-FU) on activated lymphocytes was explored. MATERIALS AND METHODS: The in vitro effects of 5-FU on DNA synthesis in mitogen-activated lymphocytes from healthy volunteers were compared to those of the antimetabolites doxorubicin, cyclophosphamide and 6-mercaptopurine. These effects were assessed by alterations in the phenotypic profile and the percentage of cells in various phases of the cell cycle, as well as by the secretion of T helper (Th)1 and Th2 cytokines (ELISA). RESULTS: Unlike 5-FU, the other antimetabolites failed to augment DNA synthesis in activated lymphocytes. The effect of 5-FU correlated with an increase in the percentage of cells in the S-phase and caused an increased in CD4+ cells, a decrease in CD56+ cells and a shift of the cytokine secretion pattern from Th2 to Th1. CONCLUSION: 5-FU exhibited a unique effect on DNA synthesis in activated lymphocytes which was accompanied by selective effects on various lymphocyte subpopulations.

In vitro modulation of interleukin-2-mediated human peripheral mononuclear cell proliferation and antitumor cytotoxicity by 5-fluorouracil.

BACKGROUND: Studies on cancer patients undergoing chemotherapy have shown inhibitory effects of anticancer drugs on certain immune activities. MATERIALS AND METHODS: The in vitro effect of 5-(FU) fluorouracil was studied on both lymphocyte proliferation and natural killer (NK) cell antitumor cytotoxicity following incubation of peripheral mononuclear cells (PBMCs) from healthy donors with interleukin (IL)-2, phytohemagglutinin A (PHA) and pokeweed mitogen (PWM). RESULTS: Activation of PBMCs with IL-2, PHA and PWM in the presence of 250 and 2500 microM of 5-FU caused a marked decrease in both the induction of activated natural killer (ANK) cell cytotoxic activity and DNA synthesis, while 2.5 microM increased DNA synthesis by 195%, 58% and 222% for IL-2, PHA and PWM, respectively, more than cells cultured without the drug. No effect of 5-FU was noted on mature ANK cells. CONCLUSION: 5-FU exhibits diverse effects on lymphocyte proliferation and on the generation of ANK antitumor cytotoxic activity.

Inhibition of lymphokine-activated killer cells generation in vitro by soluble factors released from mixed human tumor and peripheral blood mononuclear adherent cells culture.

BACKGROUND: A variety of molecules produced by both tumor cells and normal cells reduce the activity of lymphokine-activated killer (LAK) cells. We tested the possible cross-regulation of mel-624 melanoma cells and adherent peripheral blood mononuclear cells (PBMCs) in affecting LAK cell activity. METHODS: PBMC adherent cells were cultured together with mel-624 melanoma cells. Supernatant was transferred to a 4-day LAK cells generation culture consisted of PBMC nonadherent cells and interleukin-2. LAK cytotoxic activity was tested in a 4-hour assay against Daudi tumor cells prelabeled with sodium (51)chromate. RESULTS: The supernatant produced within the first 48 hours of mixed mel-624 melanoma cell and adherent PBMC culture substantially (by 69 percent) reduced the generation of LAK cells, whereas the supernatant from either tumor culture or adherent PBMC culture had no effect. The inhibitory effect was manifested on the generation of LAK cells when autologous nonadherent cells were cultured with 1,000 units/ml IL-2, but there was no effect on mature LAK cell cytotoxic activity. Inhibition of LAK cell generation was partially dependent on protein synthesis and was not mediated by transforming growth factor ss (TGF-ss). CONCLUSION: Our results point toward the production of soluble, yet unidentified proteins, in mixed tumor-adherent PBMC cultures, which substantially reduced the induction of LAK cells in culture.

Phenotype and function of lymphocytes from the neonatal umbilical cord compared to paired maternal peripheral blood cells isolated during delivery.

In the present study, we analyzed the immunological characteristics of mononuclear cells (MNC) isolated from both neonatal umbilical cord blood (UCB) and maternal peripheral blood (MPB) during the delivery. The in vitro proliferative response of UCB T lymphocytes was significantly reduced compared to the maternal response to phytohemagglutinin A, pokeweed mitogen, and alloantigen stimulation, in correlation with the lower percentage of UCB than MPB lymphocytes, but not with that of B cells. The mean cytotoxic activity level of interleukin-2 (IL-2)-activated natural killer (NK) was higher in UCB than in MBP, whereas the percentage of CD56(+) NK cell count was similar. Our results show differences in the immune reactivity of T and B lymphocytes from neonate and adult isolated under similar physiological conditions.

Immunohistochemistry Evaluation of the Effect in Vivo of Tumor Necrosis Factor (TNF)-alpha on Blood Vessel Density in Murine Fibrosarcoma.

PURPOSE: Angiogenesis is essential for tumor growth and metastases, thus bestowing obvious importance upon methodologies which could enable its inhibition. MATERIALS: C57BL/6 female mice bearing a subcutaneous (s.c.) MCA205 fibrosarcoma were used. METHODS: Ten mice were divided equally into two groups. One group was injected intraperitoneally (i.p.) with 10 mug tumor necrosis factor-alpha (TNF-alpha and the other (controls) with Hanks balanced salt solution (HBSS). Tumor growth was monitored at least twice weekly. The number of endothelial cells in the blood microvessels was assessed by immunohistostaining on paraffin-embedded tumor tissue sections using vascular endothelial growth factor (VEGF) and Factor 8 antibodies. Expression of the p53 gene was similarly assessed by immunohistostaining. RESULTS: Injection of 10 mug TNF-alpha into the tumor-bearing mice reduced the number of endothelial cells in the blood microvessels by 46% on day 3 post-injection which was accompanied by an increase (by 37%) in the expression of p53 in these cells. It also inhibited tumor growth compared to the HBSS-injected group starting at 17 days post-cytokine injection. DISCUSSION: The antitumor in vivo effect exerted by TNF-alpha on established murine sarcoma s.c. tumors may be due to an earlier effect of the cytokine on the tumor's blood microvessels, probably through an apoptotic mechanism involving the p53 gene.

Localization of a specific germ cell marker, DAZL1, in testicular germ cell neoplasias.

DAZ-like 1( DAZL1) is a germ cell-specific protein expressed in both male and female gonads. The DAZL1 gene, which maps to chromosome 3 in humans, is an autosomal homologue to the Deleted in AZoospermia ( DAZ) gene(s) located on the Y chromosome. We studied the expression of DAZL1 by means of immunohistochemistry in order to determine its distribution among testicular germ cell neoplasias and among the intratubular lesions in their vicinity. Our results demonstrated that expression of DAZL1 protein was consistently observed in scattered cells in all intratubular germ cell neoplasias (IGCN) of the unclassified type, as well as in some of the intratubular seminomas. Foci of DAZL1 immunopositive cells were detected in pure seminomas, while single immunopositive cells were dispersed in the seminomatous component of mixed germ cell neoplasias. All the nonseminomatous components were negative for DAZL1 expression. These findings demonstrate an antigenic heterogeneity of seminoma cells. The localization of a specific germ cell protein, DAZL1, in the putative ontogenic progenitor, IGCN, and in their putative derivative, seminoma, provides further support for the hypothesis that IGCN is the precursor of germ cell neoplasias.