TGF-{beta} signaling of human T cells is modulated by the ancillary TGF-{beta} receptor endoglin

Transforming growth factor beta (TGF-beta) inhibits T cell activation and alters differentiation of naive T cells into effector cells. Although four main cell-surface proteins can interact with TGF-beta, only the signaling receptors type I (TGF-betaR type I) and type II (TGF-betaR type II) have so far been described on T cells. The aim of the present study was to investigate the expression of the ancillary receptor endoglin (CD105) by T cells and its role in TGF-beta-mediated signal transduction and function. CD105 expression was analyzed on resting and activated human CD4(+) T cells by flow cytometry, western blot, immunoprecipitation, proliferation and SMAD-responsive reporter gene assays. CD4(+) T cells constitutively expressed CD105 in memory T cells and partially also in naive T cells; however, surface expression is regulated and is increased following TCR engagement, which induced serine/threonine phosphorylation of CD105. In contrast to the suppressive signal mediated by the TGF-beta, cross-linking of CD105 substantially enhanced T cell proliferation, indicating that CD105 by itself mediates signal transduction. Furthermore, CD105 cross-linking induced SMAD-independent signaling via ERK kinase phosphorylation. The present study demonstrates that CD105 is expressed on the surface by activated CD4(+) T cells and CD3 regulated by post-translational means. Furthermore, CD105 acts as a regulatory receptor, counteracting TGF-beta-mediated suppression.     

Distinct binding specificities of integrins {alpha}4{beta}7 (LPAM-1), {alpha}4{beta}1 (VLA-4), and {alpha}IEL{beta}7

Integrin receptors are Important for regulating lymphocyte reclrculatlonand recruitment to sites of inflammation. Transfoctants of theB cell lymphoma 38C13 were generated that differ exclusivelyin the expression of integrin ß1 or ß7 subunltsallowing for a functional comparison of lymphocyte Peyer's patchHEV adhesion molecule 1 (LPAM-1) (4ß7) and very lateantigen 4 (VLA-4) (4ß1) in an Identical cellular environment.Whereas 38-ß7 transfectants bound to purified andcellular mucosal addressin cell adhesion molecule (MAdCAM-1),unstlmulated 38-ß1 cells failed to bind MAdCAM-1.Treatment of 38-ß1 cells with Mn²⁺ but not with PMAinduced low level binding to MAdCAM-1. MAdCAM-1 adhesion of38-ß7 cells was constitutive and not enhanced by Mn²⁺treatment. Similarly, MAdCAM-1-dependent adhesion to mucosalhigh endothellal venules was shown for 38-ß7 but notfor 38-ß1 cells. The results therefore establish theLPAM-1 - MAdCAM-1 Interaction as the functionally dominant adhesionpathway for regulating lymphocyte homing to mucosal sites. Nonetheless,the activated VLA-4 on some lymphocytes may be involved in MAdCAM-1recognition or promote binding to MAdCAM-1 In other tissues.By contrast, 38-ß7 and 38-ß1 transfectantsdid not differ in their binding capacity for vascular cell adhesionmolecule 1 (VCAM-1) or fibronectin and LPAM-1 did not displayany preference for interacting with either MAdCAM-1 or VCAM-1.LPAM-1 may therefore contribute significantly to cellular functionspreviously attributed to VLA-4. Interestingly, functional analysisof the intraepithellal lymphocyte integrin IELß7 whichIs structurally related to LPAM-1 did not reveal detectablebinding activity for MAdCAM-1, VCAM-1, or fibronectin.     

The expression of {alpha}V, {alpha}5, {beta}1, and {beta}3 integrin chains on ejaculated human spermatozoa varies with their functional state

Evidence has been presented suggesting the involvement of integrinsand their ligands in mammalian fertilization. In this studywe asked whether the     

Estrogen receptor beta is expressed in human colorectal adenocarcinoma

Estrogen receptor beta (ER-beta) has recently been detected in a human colon cancer cell line. The aim of this work was to determine whether ER-beta is expressed in human colorectal carcinoma (CRC) tissue and the extent of this expression. ER-beta expression in CRC was investigated by immunohistochemical staining of sections of formalin-fixed, paraffin-embedded tissue from 55 CRC. The percent of positive cells was recorded. ER-beta immunoreactivity was always present in normal epithelium and adenomas in the same sections of some CRC and was always nuclear. In CRC, nuclear ER-beta immunoreactivity was detected in >10% of the cancer cells in 67% of the cases and was almost always associated with cytoplasmic immunoreactivity. There were no statistically significant differences between the ER-beta-positive and -negative groups in regard to depth of invasion, nodal metastases, or survival, regardless of the cut-off value used. We conclude that (1) a significant number of CRCs are positive for ER-beta. (2) estrogen may play an important role in the proliferation of normal colonic epithelium, and (3) there is differential localization of ER-beta immunoreactivity between normal colon, adenomas, and CRCs. Whether different ER-beta isoforms are differentially expressed in CRCs, and whether human CRCs respond to treatment with antiestrogens, is the subject of studies currently in progress.     

The role of pregnancy-specific {beta}-1 glycoprotein (SP1) in assessing human blastocyst quality in vitro

The advent of new culture techniques resulting in more than60% of embryos developing in vitro to the blastocyst stage suggeststhat blastocyst transfer in humans with its potential to increasein-vitro fertilization success rates could be a feasible option.Blastocyst quality markers, however, are required to ensurethat an increase in numbers is not achieved at the expense oflowering quality. We have previously reported a morphology basedmethod for grading blastocysts. The current study sought todetermine whether the secretion of pregnancy-specific -1-glycoprotein(SP1) (a trophoblast product known to be associated with fetalwell-being) by blastocysts of differing quality was reflectedin the morphological grading. SP1 concentrations were measuredin the culture medium of grade 1 (n=19), grade 2 (n=4) and grade3 (n=4) blastocysts as well as vacuolated morulae (n=6) dailyfrom day 1 to day 14. Cumulative SP1 concentrations secretedby blastocysts were significantly higher than those secretedby vacuolated morulae. However, SP1 levels could not distinguishbetween blastocysts of differing morphological grades. The inconsistentpattern of secretion suggests that at this early stage of development,SP1 secretion is probably not sufficiently defined to allowany differences in levels to be reflected by the morphologicalgrading.     

Interleukin-1{alpha} and -{beta} modulation of luteinized human granulosa cell oestrogen and progesterone biosynthesis

This study was designed to test the hypothesis that inter-leukin-1(IL-1) and directly affect progesterone, and oestradiol productionin cultures of purified human granulosa cells. Luteinized granulosacells were obtained from women during in-vitro fertilizationcycles. Granulosa cells with and without associated white bloodcells were cultured in the presence of IL-1 and IL-1 (0.5–50ng/ml) for 48 h. Media were changed at 24 h intervals and assayedfor progesterone and oestradiol. In separate experiments, granulosacell viability was assessed with the tetrazolium salt reductionassay, haemocytometer cell counts, and Trypan blue dye exclusion.Our results indicate that progesterone synthesis by basal andhuman chorionic gonadotrophin (HCG)-stimulated granulosa cellsco-cultured with white blood cells was inhibited by 5.0 ng/mlof IL-1 and IL-1 at 48 h of culture. In the presence of whiteblood cells, granulosa cell oestradiol synthesis was inhibitedby IL-1 but not IL-1. Oestradiol was inhibited after both 24and 48 h of culture and was maximally affected by 5.0 ng/mlof IL-1. In contrast, basal and HCG-stimulated oestradiol productionby granulosa cells cultured free of white blood cells was inhibitedonly by IL-1. IL-1 at 5.0 ng/ml produced maximal inhibitionof basal oestradiol (57%) and HCG-stimulated oestradiol (41%)production at 48 h of culture. Gonadal steroid inhibition byIL-1 and IL-1 was not mediated through cytotoxic or antiproliferativeeffects on granulosa cells. Specificity of the granulosa cellresponse to IL-1 and IL-1 was demonstrated by abrogation ofsteroid inhibition with anti-IL-1 and IL-1 neutralizing antibodies.In conclusion, IL-1 directly inhibited the production of oestradiolby human ovarian granulosa cells. IL-1 and IL-1 also exertedindirect effects on steroid production via white blood cellsthat are usually present in granulosa cell cultures if stepsare not taken to remove them. These data support the hypothesisthat cytokines play an important role in intra-ovarian regulationof steroid biosynthesis.     

Concentration of interleukin-1{beta} correlates with prostaglandin E2 and F2{alpha} in human pre-ovulatory follicular fluid

To investigate the role(s) of interleukin-1 (IL-1) in humanovarian function, we measured the concentrations of IL–1,prostaglandins (PGs) and steroids in follicular fluid of 90stimulated ovaries, with reference to oocyte maturation. Concentrationsof IL-1 were significantly higher in the follicles from whichmature oocytes were recovered than in follicles from which oocytescould not be recovered (P < 0.05). IL-1 concentrations alsoincreased in association with oocyte maturation. Positive significantcorrelations were seen between IL-1 and prostaglandin E2 (PGE2)(r = 0.47, P < 0.001), and between IL-1 and prostaglandinF₂ (PGF₂) (r = 0.22, P < 0.05) in pre-ovulatory follicularfluid, but not between IL–1 and oestradiol, or betweenIL-1 and oestradiol, or between IL-1 and progesterone. 0Follicularfluid IL-1 might contribute to prostaglandin-induced oocytematuration and ovulation.     

The cloning and expression of a sodium channel {beta}1-subunit cDNA from human brain

Electrical excitability of neurons and muscle cells is mediatedlargely through the actions of the voltage-gated sodium channel.Initiation and propagation of the action potential is a directresult of the precisely controlled inward flux of sodium throughthese channels. Much attention has been paid to the sodium channel     

Non-random employment of V{beta}6 and J{beta} gene elements and conserved amino acid usage profiles in CDR3 regions of human fetal and adult TCR {beta} chain rearrangements

We have studied the usage of V_ß36, D_ß, andJ_ß elements, and the composition of the CDR3 regionsof human fetal TCR ß chain rearrangements In a 17week old fetal thymus and In fetal liver, bone marrow, spleen,and cord blood at 11 and 13 weeks of gestation. These fetalsequences were compared with TCR ß chain rearrangementsobtained from post-partum thymus, adult spleen, and adult peripheralblood mononuclear cells. Both fetal and adult TCR V_ß6rearrangements exhibited a non-random usage of V_ßand J_ß elements. Up to 90% of the sequences obtainedat 11 weeks of gestation used J_ß2 elements, most notablyJ_ß2.1. In the 13 and 17 week old fetal and in theadult tissues, J_ß1 elements were used In 30% of therearrangements while, within the J_ß2 rearrangements,J_ß2.1 and J_ß2.7 were used most frequently.Both fetal and adult TCR ß chain CDR3 regions showednon-random usage of amlno acids. However, the early fetal repertoirewas further limited due to the relative absence of N-reglonsin up to 60% of the 11 and 13 week old TCR ß chainrearrangements, resulting In smaller antigen binding sites.In fetal and adult TCR ß chain rearrangements thedistribution patterns of the length of N-regions and the usageprofiles of J_ß elements were similar in hematopoletlcand peripheral organs, suggesting no apparent preference forparticular TCR ß chain rearrangements. On the whole,the data showed that both the available fetal and adult TCRV_ß6 repertoires are seemingly smaller than expectedon the basis of random usage of V_ß and J_ßelements and amlno acid composition of the CDR3 regions.     

Gestational development of human placental 17{beta}-hydroxysteroid oxidoreductase types 1 and 2

Human placenta is a rich source of 17-hydroxysteroid oxidoreductase(17-HOR) type 1, a cytosolic enzyme highly specific for 17-oestradiol,and type 2, a microsomal form reactive with both oestradioland testosterone. Although a number of studies have establishedthat 17-HOR activity is present in placenta as early as weeks4–5 of gestation, more specific data on the pattern ofdevelopment of these two enzyme forms are lacking. In this study,samples of villous tissue from weeks 7–20 of gestationwere fractionated into cytosol and microsomes and 17-HOR activityassayed under conditions which differentiate between the twoenzyme types. Type 1 activity with oestradiol of cytosol andmicrosomal type 2 activity with oestradiol and testosteroneincreased from week 7 to week 20. Activities at 17–20weeks approximated those at 38dash;40 weeks. The high, cytosolicoestradiol/T activity ratio (160 ± 20), characteristicof 17-HOR type 1, was constant between weeks 7 and 20, as wasthe low microsomal ratio (3.4 ± 0.4) characteristic ofthe type 2 activity. There was a relationship between cytosolictype 1 activity and microsomal type 2 activity between weeks7 and 20 (r equals; 0.59, P equals; 0.0055). These results indicateboth activities increase coincident with the luteal-placentalshift and that their temporal patterns of development are relatedbetween weeks 7 and 20 of gestation.     

Effects of progesterone and oestradiol-17{beta} on 17{beta}-ol-dehydrogenase activity in stromal cells of human endometrium under in-vitro conditions

A 17-ol-dehydrogenase activity could be demonstrated in fibroblastmonolayer cell cultures of proliferative human endometrium.After 24 h incubation 100 nCi [6, 7–3H] oestradiol-17was completely oxidized to oestrone. Progesterone was not ableto enhance the metabolizing velocity. In contrast, progesteroneincubation revealed a decreasing oxidation rate with increasingmolarity. Histological changes after transformation of the endometriumare discussed to explain in-vivo results showing an increased17-ol-dehydrogenase activity in the secretory phase of the cycle     

Andrology: Evidence of {beta}1 integrins and fibronectin on spermatogenic cells in human testis

Interactions between human spermatozoa and oocytes are an essentialevent in the process of fertilization. Cell —cell andcell — matrix interactions in somatic cells are mediatedby adhesion molecules such as 1 integrins (very late antigens;VLA). Therefore, we investigated the expression of 1 integrinsand the matrix proteins collagen IV, fibronectin and lamininin human testis by immunohistology. Monoclonal antibodies againstthe chain of 1 integrins reacted with the basement membraneof the tubuli seminiferi, spermatocytes, spermatids and testicularspermatozoa. The 3, –5 and –6 chains of 1 integrinsshowed the same pattern, whereas, the 1, –2 and –4chains could not be detected on spermatogenic cells. These VLAsubunits were localized on endothelial cells, leukocytes andbasement membranes. Matrix proteins such as laminin, collagenIV and fibronectin were detectable as components of basementmembranes in human testis. Germinal cells except spermatogoniaexpressed fibronectin only. These results demonstrate that 1integrins and matrix proteins in human testis are normally expressedon somatic tissue and that germinal cells, especially spermatocytes,spermatids and spermatozoa show positive reactions with antibodiesagainst the VLA–3, –5 and –6 complexes andfibronectin. These findings suggest a production of B1 integrinsand fibronectin during the spermatogenesis and a role of theseproteins in adhesive mechanisms of spermatozoa similar to somaticcell—cell or cell—matrix interactions.     

Serum protects against azurophil granule dependent down-regulation of complement receptor type 1 (CR1) on human neutrophils

We have investigated the effect of gradual degranulation on the expression of functional receptors (CR1 and CR3) on human neutrophils. Incubation with increasing concentrations of fMLP (10^–10–10^–7M) translocated CR1 and CR3 to the cell surface in a similar kinetic pattern. When reaching maximal expression of receptors (10^–7 M fMLP), 78 ± 10% and 87 ± 9% of the total pool of CR1 and CR3, respectively, were translocated to the cell surface. To drive the mobilization process further, cytochalasin B was introduced to increase the stimulatory effect of fMLP. No further increase in CR1 surface expression was obtained. However, we found a characteristic time course of surface appearance of CR1 and CR3 with a maximal surface expression within 1 minute, followed by a time-related down-regulation of CR1 but not CR3. In addition, the total pool of CR1 in cytochalasin B treated neutrophils was reduced after 15 minutes stimulation with fMLP measured by flow cytometry and immunoblotting, indicating degradation of CR1. The down-regulation of CR1 was concomitant with a translocation of azurophil granules, in terms of upregulation of CD63. Azurophil, but not specific nor secretory, granule fractions caused a down-regulation of CR1 on fMLP activated neutrophils. The presence of human sera and serine protease inhibitor protected CR1 from down-regulation. Together, these findings indicate that intracellular stored proteases, released in the late part of the sequential mobilization process, alters the expression of functional receptors mobilized in the early part of the mobilization process. The findings also focus on the importance of the microenvironment for the net outcome of neutrophil activation in terms of functional receptor expression.