Increase In 3H-thymidine Incorporation Research Articles

Signalling pathways involved in the synergistic effects of human growth differentiation factor 9 and bone morphogenetic protein 15.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) act synergistically to regulate granulosa cell proliferation and steroid production in several species. Several non-Sma and mothers against decapentaplegic (SMAD) signalling pathways are involved in the action of murine and ovine GDF9 and BMP15 in combination, with the pathways utilised differing between the two species. The aims of this research were to determine if human GDF9 and BMP15 also act in a synergistic manner to stimulate granulosa cell proliferation and to identify which non-SMAD signalling pathways are activated. Human GDF9 with BMP15 (GDF9+BMP15) stimulated an increase in (3)H-thymidine incorporation (P<0.001), which was greater than the increase with BMP15 alone, while GDF9 alone had no effect. The stimulation of (3)H-thymidine incorporation by GDF9+BMP15 was reduced by the addition of inhibitors to the SMAD2/3, nuclear factor-KB (NF-KB) and c-Jun N-terminal kinase (JNK) signalling pathways. Inhibitors to the SMAD1/5/8, extracellular signal-regulated kinase mitogen-activated protein kinase (ERK-MAPK) or p38-MAPK pathways had no effect. The addition of the BMP receptor 2 (BMPR2) extracellular domain also inhibited stimulation of (3)H-thymidine incorporation by GDF9+BMP15. In conclusion, human GDF9 and BMP15 act synergistically to stimulate granulosa cell proliferation, a response that also involves species-specific non-SMAD signalling pathways.

Characterization of the in vivo function of RGC-32 in T-cells (P1151)

Abstract We have previously shown that RGC-32 is involved in cell cycle regulation in vitro. To define the in vivo role of RGC-32, we generated RGC-32 knockout mice. These mice develop normally and do not spontaneously develop overt tumors. To assess the effect of RGC-32 deficiency on cell cycle activation in T cells we determined the proliferation rate of CD4 and CD8 from spleens of RGC-32-/- mice compared to wild type (WT) mice. CD4 T cells from RGC-32-/- mice display a significant increase in 3H-thymidine incorporation when compared with WT mice after stimulation with anti-CD3/anti-CD28. In addition, both CD4 and CD8 T cells from RGC-32-/- displayed a significant increase in the proportion of proliferating Ki67+ cells, indicating that in T cells, RGC-32 has an inhibitory effect on cell cycle activation. Furthermore, Akt and FOXO1 phosphorylation induced in CD4 from RGC-32-/- were significantly higher indicating that RGC-32 inhibits cell cycle activation by suppressing FOXO1 activation. To further examine the effect of RGC-32 on T cell functions we investigated the effect of RGC-32 on T regulatory (Treg) cells differentiation. A significantly lower percentage of CD4+ FoxP3+ Treg cells was found in unstimulated cells from RGC-32-/- compared to WT mice. Significantly fewer CD4+ FoxP3+ Treg cells are induced by TGF-β in RGC-32-/- compared to WT mice. Thus, RGC-32 is involved in controlling cell cycle in vivo, and in addition seems to play a role in the differentiation of Treg cells.

Protection of immunocompromised mice from fungal infection with a thymus growth-stimulatory component from Selaginella involvens, a fern

Context: Recent studies have shown that the water extract of Selaginella involvens (Sw.) Spring, a wild fern, exhibits thymus growth-stimulatory activity in adult mice (reversal of involution of thymus) and remarkable anti-lipid peroxidation activity. Follow-up studies were carried out in the present study.Materials and Methods: Activity-guided isolation of the active component (AC) was carried out. The effect of AC on immune function was studied using fungal (Aspergillus fumigatus) challenge in cortisone-treated mice. The in vitro antifungal activity of AC was assayed using disc diffusion assay. In vitro and in vivo effect of AC on DNA synthesis in thymus was studied using 3H-thymidine incorporation. In in vitro anti-lipid peroxidation, hydroxyl radical scavenging and inhibition of superoxide production were assayed.Results: The active principle/component (AC) was isolated in a chromatographically pure form from the water extract of S. involvens. AC showed positive reaction to glycosides. AC possessed both thymus growth-stimulatory and antioxidant properties. It protected cortisone-treated mice from A. fumigatus challenge. It did not exhibit in vitro antifungal activity. Increased 3H-thymidine incorporation was observed in the reticuloepithelium of thymus obtained from AC-treated mice. However, in vitro AC treatment to thymus for 5 h did not result in an increase in 3H-thymidine incorporation.Discussion and conclusion: AC (named as Selagin), from S. involvens, could reverse involution of thymus to a large extent, exhibit remarkable antioxidant activity, and protect immunocompromised mice from fungal infection. Therefore, it is very promising for the development of a drug to ameliorate old age-related health problems and prolong lifespan.

Urotensin II-induced signaling involved in proliferation of vascular smooth muscle cells

The urotensin II receptor, bound by the ligand urotensin II, generates second messengers, ie, inositol triphosphate and diacylglycerol, which stimulate the subsequent release of calcium (Ca(2+)) in vascular smooth muscle cells. Ca(2+) influx leads to the activation of Ca(2+)-dependent kinases (CaMK) via calmodulin binding, resulting in cellular proliferation. We hypothesize that urotensin II signaling in pulmonary arterial vascular smooth muscle cells (Pac1) and primary aortic vascular smooth muscle cells (PAVSMC) results in phosphorylation of Ca(2+)/calmodulin-dependent kinases leading to cellular proliferation. Exposure of Pac1 cultures to urotensin II increased intracellular Ca(2+), subsequently activating Ca(2+)/calmodulin-dependent kinase kinase (CaMKK), and Ca(2+)/calmodulin-dependent kinase Type I (CaMKI), extracellular signal-regulated kinase (ERK 1/2), and protein kinase D. Treatment of Pac1 and PAVSMC with urotensin II increased proliferation as measured by (3)H-thymidine uptake. The urotensin II-induced increase in (3)H-thymidine incorporation was inhibited by a CaMKK inhibitor. Taken together, our results demonstrate that urotensin II stimulation of smooth muscle cells leads to a Ca(2+)/calmodulin-dependent kinase-mediated increase in cellular proliferation.

Urotensin II Induced Signal Involved in Proliferation of Vascular Smooth Muscle Cells

Urotensin II receptor (UIIR), bound by the ligand Urotensin II (UII), generates second messengers IP3 and DAG, which stimulates the subsequent release of calcium (Ca2+) in vascular smooth muscle cells (VSMC). Ca2+ influx leads to the activation of Ca2+ dependent kinases via calmodulin binding, resulting in cellular proliferation. We hypothesize that UII signaling in pulmonary arterial vascular smooth muscle cells (Pac1) and primary aortic vascular smooth muscle cells (PAVSMC) results in phosphorylation of Ca2+/ CaM dependent kinases leading to cellular proliferation. UII exposure to Pac1 cell cultures increased intracellular Ca2+, which subsequently activated Ca2+/CaM dependent kinases (CaMKK &amp; CaMKI), ERK 1/ 2, and PKD (PKC‐µ). Pac1 and PAVSMC cells treated with UII resulted in increased proliferation as measured by 3H thymidine uptake. UII‐induced increase of 3H thymidine incorporation was inhibited by a CaMKK inhibitor. Taken together, our results demonstrate that UII stimulation of smooth muscle cells leads to cellular proliferation, utilizing signaling mechanisms involving Ca2+/CaM dependent kinases.

134. EPIDERMAL GROWTH FACTOR RECEPTOR/MAPK3/1 PATHWAY CROSS-TALK ENABLES GROWTH DIFFERENTIATION FACTOR 9 TO SIGNAL THROUGH SMAD2/3 IN MOUSE GRANULOSA CELLS

Oocyte-secreted growth differentiation factor 9 (GDF9) plays a critical role throughout folliculogenesis. It has been shown to control many functions of granulosa cells, including gene expression, steroidogenesis and proliferation. This study investigates the cellular requirements that allow GDF9 to act on granulosa cells. Our results showed that GDF9 (20 ng/ml)-stimulated mouse granulosa cells 3H-thymidine incorporation was inhibited by a type 1 receptor Alk4/5/7 inhibitor (SB431542, 5 μM), by an epidermal growth factor (EGF) receptor inhibitor (AG1478, 5μM) and a MEK1 inhibitor (U0126, 10 μM). Interestingly, activin A- and TGFβ-stimulated 3H-thymidine incorporation shared similar inhibitor sensitivity. Moreover, when denuded oocytes were used as the mitogenic agent, SB431542, AG1478 and U0126 all prevented the increase in 3H-thymidine incorporation. Oocyte-stimulated 3H-thymidine incorporation in secondary follicles and cumulus-oocyte complexes were also sensitive to Alk4/5/7, EGF receptor and MEK1 inhibition. Basal and EGF-stimulated levels of phopho-MAPK3/1 were inhibited by using the EGF receptor inhibitor, but were not affected by inhibition of Alk4/5/7 or by adding GDF9 in granulosa cells. Using granulosa cells transfected with a SMAD3-luciferase reporter construct, GDF9-stimulated SMAD3 response could be inhibited by Alk4/5/7, EGFR and MEK1 inhibitors. Genes involved in cumulus cells expansion (Ptx3 and Has2) were upregulated in granulosa cells by co-culturing with denuded oocytes and that upregulation was inhibited by Alk4/5/7 as well as by EGF receptor inhibition. These results suggest that TGFβ superfamily members signalling through Smad2/3 share a common requirement of EGF receptor-dependant phospho-MAPK3/1 throughout folliculogenesis. These results strongly suggest that, apart from its role in the transmission of the ovulatory LH signal within the ovarian follicle, EGF receptor pathway might serve as modulators of GDF9 action on granulosa cells. Hence the interaction between endocrine and oocyte signalling may be mediated at the level of MAPK and Smad2/3 cross-talk in granulosa cells.

Cyclosporine Promotes Epstein-Barr Virus-Infected Human B-Cell Transformation Assayed by Three Correlated Assay Methods

We have reported that cyclosporine (CsA) has direct effect to promote Epstein-Barr virus (EBV) transformation of human peripheral blood B lymphocytes. In this article, we have reported that CsA promoted EBV-infected, human B-cell transformation as assayed by three methods of colony number counting, cell number counting, and 3H-thymidine incorporation. At first, we sought to correlate the three methods in EBV-infected human B-cell transformation, observing that they are convenient correlate with each other, and only vary in the degree when transformed cells are compared to the controls. Based on these pilot experiments, the three assay methods were then applied to CsA-treated and nontreated, EBV-infected human B cells to investigate whether CsA treatment promoted EBV-infected human B-cell transformation. We observed that CsA treatment increased colony formation above the control value of 28 ± 4.5/well to 49 ± 4.3 (colonies/well; n = 5; P < .05). CsA treatment increased the cell number from the control of 33,025 ± 1900 to 50,925 ± 4194 (cells/well; n = 5; P < .05). CsA treatment increased 3H-thymidine incorporation from the control result of 12,481 ± 1341 to 26,514 ± 5464 (CPM/well; n = 5; P < .05). In conclusion, CsA promoted EBV-B-cell transformation in three correlated assay methods in vitro using a model of posttransplant lymphoproliferative disorder.

Vitreous humor and albumin augment the proliferation of cultured retinal precursor cells

Intravitreal injection is an important delivery route for studies involving the transplantation of various types of precursor cells to the retina; however, the effect on these cells of exposure to the vitreous microenvironment has not been specifically investigated. Here vitreous humor was evaluated for the potential to influence the proliferation of rat retinal precursor cells in vitro. Cells were isolated at embryonic day 19 and plated in standard proliferation medium in the presence or absence of fluid expressed from porcine vitreous humor. Cellular proliferation at different concentrations of vitreous fluid supplementation was quantified by using a (3)H-thymidine incorporation assay. Active components of vitreous fluid were partially characterized by gel filtration chromatography (GFC) and UV spectral analysis. The effect of each vitreous fraction on proliferation was determined as well. Results showed that addition of 20% vitreous fluid to primary rat retinal cultures significantly increased (3)H-thymidine incorporation compared with growth medium without vitreous supplementation. A vitreous fraction showing growth-promoting activity was localized to a molecular mass range <1000 Da, consistent with ascorbic acid. Ascorbic acid was confirmed in vitreous fluid by UV spectral analysis. Growth-augmenting activity was present in higher molecular mass vitreous fractions, consistent with protein components. Albumin, the major protein in vitreous fluid, was found to augment proliferation. Because vitreous-associated augmentation of retinal precursor proliferation remains an epidermal growth factor-dependent phenomenon, the proliferative status of transplanted cells in the vitreous cavity is likely determined by a combination of factors.

Surface expression of adenosine deaminase in mitogen-stimulated lymphocytes.

Adenosine deaminase (ADA) expression on the surface of mitogen-stimulated lymphocytes was studied by flow cytometry. The gate for lymphocytes was located by cell size (forward scatter), cytoplasmic complexity (side scatter) and by expression of the markers CD2, CD4, CD8 and CD19. After mitogenic proliferation two populations appeared, one corresponding to non-stimulated cells, and the other consisting of larger cells which showed relatively high expression of adenosine deaminase on their surface. The increase was similar to that observed for CD71 expression, and paralleled the increase in 3H-thymidine incorporation. There was a correlation between ADA and CD71 expression (r = 0.92 for phytohaemagglutinin (PHA) and 0.97 for pokeweed mitogen (PWM)). These results suggest a role for ecto-adenosine deaminase in lymphocyte proliferation and/or triggering.

Angiotensin II–Induced Proliferation of Neonatal and Adult Rat Cardiac Fibroblasts

To the Editor: Olson et al1 reported that stimulation of adult rat cardiac fibroblasts with angiotensin II (Ang II) resulted in a significant increase in 3H-thymidine incorporation over basal. Ang II indeed stimulated the proliferation of neonatal rat cardiac fibroblasts.2 However, discordant findings …

Anti-diabetic properties of the Canadian lowbush blueberry Vaccinium angustifolium Ait.

Incidence of type II diabetes is rapidly increasing worldwide. In order to identify complementary or alternative approaches to existing medications, we studied anti-diabetic properties of Vaccinium angustifolium Ait., a natural health product recommended for diabetes treatment in Canada. Ethanol extracts of root, stem, leaf, and fruit were tested at 12.5 microg/ml for anti-diabetic activity in peripheral tissues and pancreatic beta cells using a variety of cell-based bioassays. Specifically, we assessed: (1) deoxyglucose uptake in differentiated C2C12 muscle cells and 3T3-L1 adipocytes; (2) glucose-stimulated insulin secretion (GSIS) in beta TC-tet pancreatic beta cells; (3) beta cell proliferation in beta TC-tet cells; (4) lipid accumulation in differentiating 3T3-L1 cells; (5) protection against glucose toxicity in PC12 cells. Root, stem, and leaf extracts significantly enhanced glucose transport in C2C12 cells by 15-25% in presence and absence of insulin after 20 h of incubation; no enhancement resulted from a 1 h exposure. In 3T3 cells, only the root and stem extracts enhanced uptake, and this effect was greater after 1 h than after 20 h; uptake was increased by up to 75% in absence of insulin. GSIS was potentiated by a small amount in growth-arrested beta TC-tet cells incubated overnight with leaf or stem extract. However, fruit extracts were found to increase 3H-thymidine incorporation in replicating beta TC-tet cells by 2.8-fold. Lipid accumulation in differentiating 3T3-L1 cells was accelerated by root, stem, and leaf extracts by as much as 6.5-fold by the end of a 6-day period. Stem, leaf, and fruit extracts reduced apoptosis by 20-33% in PC12 cells exposed to elevated glucose for 96 h. These results demonstrate that V. angustifolium contains active principles with insulin-like and glitazone-like properties, while conferring protection against glucose toxicity. Enhancement of proliferation in beta cells may represent another potential anti-diabetic property. Extracts of the Canadian blueberry thus show promise for use as a complementary anti-diabetic therapy.

Overexpression of a truncated TrkB isoform increases the proliferation of neural progenitors

The truncated isoform of TrkB, TrkB.T1, has been shown to be expressed in the neurogenic region of rodent brain. TrkB.T1 lacks tyrosine kinase activity and it may modify the action of the full-length TrkB. We show here that the full-length TrkB and TrkB.T1 are expressed at the same relative expression levels in mouse neural progenitor cell cultures. The number of neurosphere-forming progenitors was reduced and apoptosis increased in neurospheres generated from mice overexpressing TrkB.T1 when compared with wild-type neurospheres. The proliferation of the transgenic neural progenitors was increased, as indicated by the larger average diameter of spheres (140% of control), the increased cell growth in an MTT assay (137% of control) and the faster rate of 3H-thymidine incorporation (128% of control) in the transgenic cell cultures than in controls. The proliferation of neural progenitors was also increased after lentivirus-mediated TrkB.T1 overexpression. A significant increase in 3H-thymidine incorporation (119% of control) and the average diameter of spheres (112% of control) in the TrkB.T1-transduced neurospheres compared with neurospheres transduced with the control vectors confirmed the role of TrkB.T1 in proliferation of neural progenitor. When induced to differentiate, progenitors overexpressing TrkB.T1 generated two to three times more neurons than did wild-type cells. The increase in the number of neurons correlated with an increase in the number of apoptotic cells (two-fold) at these time points. The data indicate that changes in the relative expression levels of different TrkB isoforms influence the replicative capacity of neural progenitors.

Evaluation of anex vivo murine local lymph node assay: multiple endpoint comparison

The local lymph node assay (LLNA) is used to assess the skin sensitization potential of chemicals. In the standard assay, mice are treated topically on the dorsum of both ears with test substance for 3 days. Following 2 days of rest, the initiation of the hypersensitivity response is evaluated by injecting (3)H-thymidine into a tail vein, and then measuring the levels of radioisotope incorporated into the DNA of lymph node cells draining the ears. In the current study, BALB/c mice were treated with the contact sensitizers hexylcinnamic aldehyde (HCA) and oxazolone, and the nonsensitizer methyl salicylate. The proliferative response of lymph node cells was evaluated in an ex vivo assay, in which isolated cells were cultured in vitro with (3)H-thymidine. Treatment of mice with HCA at 5-50% resulted in concentration-related increases in (3)H-thymidine incorporation, with stimulation indices ranging from 3 to 14. Low animal-to-animal variability was seen in three replicate assays testing HCA at 25%. As anticipated, the proliferative response induced by the potent sensitizer oxazolone at 0.25% was greater than HCA at all concentrations tested. Stimulation indices of 1.5 and 3 were seen in two independent experiments with methyl salicylate. These equivocal findings were likely due to the irritancy properties of the compound. Importantly, measuring ex vivo (3)H-thymidine incorporation was more sensitive than evaluating lymph node weight and cellularity, and in vitro bromodeoxyuridine incorporation. Furthermore, the results of the ex vivo LLNA were comparable to the standard assay. This study provided evidence that supports the use of an ex vivo LLNA for hazard assessment of contact hypersensitivity.

Renin, Prorenin and the Putative (Pro)renin Receptor

Renin is an aspartic protease that consists of 2 homologous lobes. The cleft in between contains the active site with 2 catalytic aspartic residues.1 Unlike other aspartic proteases such as pepsin or cathepsin D, renin is monospecific and only cleaves angiotensinogen, to generate angiotensin (Ang) I. Ang I is the precursor of the active end-product of the renin-angiotensin system (RAS), Ang II. Renin has also been called active renin to underline that an enzymatically inactive form of renin exists. In 1971, Lumbers found that amniotic fluid, left at low pH in the cold, acquired renin activity.2 Later, Skinner described a similar phenomenon in plasma.3 Acidification was not strictly necessary for this increase in Ang I-generating activity, because incubation at low temperature also increased renin activity, albeit to only 15% of activity after acidification. Soon it was postulated that this inactive, but activatable “big” renin (its molecular weight was 5 kDa higher than that of renin) was the biosynthetic precursor of renin. Hence, it was named prorenin. Only with the cloning of the renin gene in 1984 was prorenin definitively proved to be the precursor of renin.4 For reasons that are unknown, prorenin circulates in human plasma in excess to renin, sometimes at concentrations that are 100-times higher.5 Prorenin has also been demonstrated in plasma of cat, dog, cattle, pig, horse, sheep, rabbit, rat, and mouse. A 43-amino acid N-terminal propeptide explains the absence of enzymatic activity of prorenin. This propeptide covers the enzymatic cleft and obstructs access of angiotensinogen to the active site of renin. (Pro)renin is synthesized as a preprohormone. It contains a signal peptide that directs the protein to the endoplasmic reticulum and ultimately to the exterior of the cell. Both renin and prorenin can be fractionated into multiple species by isoelectric focusing. …

The influence of lidocaine and verapamil on the proliferation, CD44 expression and apoptosis behavior of human chondrocytes

Ion channels, which are responsible for the controlled functioning of many cell biological processes, are present on the cell membrane of all living human cell systems under physiological conditions. A relationship between ion channel activity and proliferation behavior has been demonstrated in various cell systems. We showed in earlier studies that there is a resting membrane potential in the cell membranes of human chondrocytes, which can be influenced by various ion channel modulators. The question is to what extent can specific modulation of the ion channel activity regulate proliferation, CD44 expression and the apoptosis behavior of human chondrocytes. Human chondrocytes were isolated from osteoarthritic knee joint cartilage. The culture was made as a monolayer in RPMI medium with the addition of 10% fetal calf serum, 50 microg/ml gentamycinsulfate and 2 microg/ml amphotericin B at 37 degrees C and 5% carbon dioxide. The voltage dependent Na+ channel blocker, lidocaine, and the calcium antagonist, verapamil, were used as ion channel modulators. Proliferation was determined using 3H-thymidine incorporation as the measure. Proof of the CD44 membrane protein was performed by flow cytometry with an FITC-conjugated CD44 antibody (anti-CD44H-FITC). For apoptosis detection, the translocation of phosphatidylserine (Annexin V-FITC assay), Apo2.7 and the Caspase activity on cytokeratin 18 were determined by flow cytometry. The results show that proliferation behavior can be regulated by lidocaine and verapamil, whereby lidocaine results in a transient increase in 3H-thymidine incorporation. Both substances resulted in marked suppression of proliferation after longer incubation times. At the same time, no influence of lidocaine could be determined on the apoptosis of human chondrocytes, whereas marked cytotoxic effects occurred under verapamil. With respect to CD44 receptor expression, incubation with lidocaine resulted in an increase of up to 43%, while suppression of up to 56% was observed with verapamil. The possibility of specific modulation of ion channel activity on the cell membrane of human chondrocytes may serve as the basis for development of new therapeutic options for the treatment of arthrosis.

Functional Separation of GVHD-Derived and Naturally Occurring CD4+CD25+ Regulatory T Cells.

Graft versus host disease (GVHD) is mediated by mature alloreactive donor T cells. Upon activation, alloreactive CD4+ T cells upregulate CD134 (OX40), a member of the TNF receptor superfamily. Using a rat hapolidentical parent into F1 model of GVHD, we recently reported that CD25 expression stratifies these CD4+134+ T cells into two alloreactive T cell subsets (Biol Blood Marrow Transplant. 2004 May;10(5):298-309; results summarized in the table below).Proliferative Response*Cytokine Secretion**Cell SubsetConcanavalin AAlloantigenIFN- γIL-10CD4+CD134+CD25−+++++++++CD4+CD134+CD25+−−+++++* Cell proliferation following stimulation with the indicated agent. Measured by 3H-thymidine incorporation. ** Cytokine secretion following stimulation with alloantigen. Neither cell subset secreted detectable levels of IL-2 or IL-4.In the current study we investigated the immune regulatory potential of the GVHD-associated CD4+CD25+ T cell subset. Cocultivation of the two GVHD-associated CD4+134+ T cell subsets revealed that the GVHD-derived CD4+CD25+ T cells suppressed, in a dose dependent manner, alloantigen-induced proliferation of the CD4+CD25− T cell subset. As this observation is similar to the suppressive activity associated with naturally occurring CD4+CD25+ regulatory T cells, cells implicated in the inhibition or suppression of a wide range of immune responses, it suggested that GVHD-associated CD4+CD25+ T cells play a regulatory role in GVHD. However, in further contrasting the biological properties of GVHD-associated CD4+CD25+ T cells with those of naturally occurring CD4+CD25+ regulatory T cells, substantive differences in these two types of regulatory T cells became apparent. At the phenotypic level, GVHD-associated regulatory T cells expressed higher levels of MHC Class II and lower levels of CD45RC and CD62L, suggesting different stages of activation. GVHD-associated and naturally occurring regulatory T cells also differed functionally. While naturally occurring regulatory T cells suppressed primary mixed lymphocyte cultures, GVHD-derived CD4+CD25+ regulatory T cells provided a potent proliferative advantage for responding T cells. When contrasted with standard mixed lymphocyte cultures, addition of GVHD-derived CD4+CD25+ regulatory T cells yielded a 15–20 fold increase in 3H-thymidine incorporation. Thus, GVHD-associated CD4+CD25+ regulatory T cells have the unique ability to differentially regulate immune responses, promoting proliferation of naive alloresponsive T cells while suppressing proliferation of previously activated GVHD-derived alloreactive CD4+CD25− T cells. These data suggest that GVHD-derived CD4+CD25+ regulatory T cells may contribute to progression of GVHD in two ways: 1) By facilitating activation of allospecific naive T cells, providing a mechanism for further amplification of the immunoinflammatory cascade associated with this disease; and 2) By maintaining a reservoir of previously activated allospecific T cells, cells that may be available for supplementation of disease-associated immunoinflammatory responses as needed.

A Novel Mechanism of Plasmin-Induced Mitogenesis in Fibroblasts.

The plasminogen activator/plasmin system plays an important role, in addition to fibrinolysis, in many pathophysiological processes. Our recent studies showed that plasmin, similar to factor VIIa/tissue factor and thrombin, upregulates Cyr61, a growth factor like immediate early response gene, in fibroblasts via protease-activated receptor-mediated signaling. In preliminary studies we found that when fibroblasts were treated with VIIa (100 nM), thrombin (10 nM) or plasmin (50 nM), in concentrations that were shown to induce Cyr61 expression maximally, VIIa and thrombin increased the DNA synthesis by only about 20–30% whereas plasmin increased the DNA synthesis by 200 to 500%. The present study was carried out to investigate potential mechanism(s) by which plasmin promotes the DNA synthesis in fibroblasts and the role of protease-activated receptors and Cyr61 in plasmin-induced DNA synthesis. The plasmin-induced DNA synthesis in fibroblasts was dependent on its protease activity as the addition of active site-inhibited plasmin had no effect; and tissue factor pathway inhibitor-2 (TFPI-2) completely abrogated the plasmin-induced response. Neutralizing antibodies against PAR-1 and not PAR-2 attenuated the plasmin-induced DNA synthesis. Cyr61 antibodies completely blocked the plasmin-induced DNA synthesis suggesting that Cyr61 acts as a down-stream mediator. Surprisingly, we found no detectable increase in Cyr61 protein in plasmin-treated cell lysates whereas Cyr61 protein levels were clearly increased in thrombin-treated cells. These data suggest that plasmin might be cleaving newly synthesized Cyr61, which accumulates in ECM. Studies performed with purified recombinant Cyr61 and tumor cells that constitutively express abundant Cyr61 provided support for this notion. To test the hypothesis that plasmin release of Cyr61 from ECM plays a role in promoting DNA synthesis, we investigated the effect of conditioned media from control, plasmin-, and thrombin-treated fibroblasts in promoting DNA synthesis. The data revealed that the conditioned media of plasmin-treated cells (the protease activity in the media was inactivated) and not others significantly increased 3H-thymidine incorporation in fibroblasts. Addition of Cyr61 antibodies to the conditioned media of plasmin-treated cells attenuated the enhancing effect of the conditioned media. In summary, the data presented herein provide a novel mechanism by which plasmin promotes cell proliferation. In this mechanism, plasmin first induces the expression of Cyr61 via activation of PAR-1 mediated signaling. Next, plasmin releases/cleaves newly synthesized Cyr61 from the ECM, making it accessible to cells. The ability of plasmin to induce mitogenesis, in addition to its pericellular proteolysis, may provide a coordinated spatiotemporal effect that is required for would healing and tissue remodeling.

4-Hydroxynonenal mediates cyclosporine promoted EBV-induced B cell transformation

Introduction. We had previously shown that cyclosporine A (CsA) directly promoted Epstein--Barr virus (EBV) infected human B cell (EBV-B cell) immortalization via oxidative stress mechanism. 4-Hydroxynonenal (HNE) is a reactive end product of lipid peroxidation. We hypothesized that HNE mediates a direct oxidative stress promoting the effect of CsA on EBV-B cell transformation. Methods. Human B cells were purified from human spleen and incubated with EBV (B95-8) overnight for infection. To assay HNE production, EBV-B cells were treated with 0, 500, and 1000 ng/ml of CsA or with HNE (positive control) or DMSO (0.1%, vehicle control) for 10 min at 37°C. HNE--Protein adducts in the cell extract was assayed by slot-blot using polyclonal HNE antibody. For 3H-thymidine uptake and LMP1 assay, EBV-B cells were treated with HNE (1 μM) for 0, 0.5, 1, and 4 h at 37°C and cultured for 2 and 4 weeks. Cells were then assayed for expression of EBV oncogene LMP1 by using PE-conjugated anti-LMP1 antibody in flow cytometry and for 3H-thymidine uptake. Results. CsA at 500 and 1000 ng/ml significantly increased the level of HNE-protein adducts in EBV-B cells over the control (arbitrary units ± SE) of 251.3 ± 7.5 to 361.3 ± 9.7 and 342.7 ± 10.7, respectively ( P < 0.05, n = 3). EBV-B cells treated with physiological concentration of HNE (1 μM) for 0.5 h and 1 h and cultured for 2 weeks had significantly increased 3H-thymidine incorporation (cpm ± SE) over a control of 839.2 ± 54 to 1192 ± 41.6 and 1998 ± 24.8, respectively ( P < 0.02, n = 6). EBV-B cells treated with HNE (1 μM) for 0.5 and 1 h and cultured for 4 weeks also had significantly increased 3H-thymidine uptake over a control of 1035 ± 206 to 2022 ± 293 and 1941 ± 229, respectively ( P < 0.03, n = 6). HNE (1 μM) treated for 4 h did not have a significant effect on cell proliferation at 2 or 4 weeks. EBV-B cells treated with HNE (1 μM) for 1 h and subsequently cultured for 2 and 4 weeks had a significantly higher (>2.0 times ) LMP1 positive cell population over the control. Conclusion. This observation provides evidence for further understanding of CsA-induced oxidative stress and direct promotion of EBV related post-transplant lymphoproliferative disorder (PTLD).

Effects of potassium and anion channel blockers on the cellular response of human osteoarthritic chondrocytes

AbstractThe aim of this article is to determine to what extent the proliferation, CD44 expression, and apoptosis behavior of cells can be influenced by the modulation of ion channel activity on the cell membrane of human osteoarthritic chondrocytes. The potassium channel blocker 4- aminopyridine (4-AP) and the chloride and anion channel blocker 4-acetamido-4′-isothiocyano-2,2′-disulfonic acid stilbene (SITS) were used as ion channel modulators. Assessment of the proliferation was done by incorporation of 3H-thymidine. The detection of apoptotic cells and expression of the hyaluronic acid binding CD44-receptor were determined by flow cytometry. The results showed that 4-AP and SITS lead to a temporary increase in 3H-thymidine incorporation, followed by a suppression of proliferation after a 12-day incubation. 4-AP causes considerable cytotoxic effects. SITS leads to necrotic cell damage. CD44 expression is increased up to 43% after incubation with 4-AP for 24 or 48 h, whereas prolonged incubation under SITS influence leads to a clear inhibition of CD44 expression. In conclusion, proliferation, CD44 expression, and apoptosis behavior of human chondrocytes can be influenced by modulation of ion channel activity. These results serve as a basis for further investigations to extend the therapeutic possibilities in the treatment of arthritis.

Glucagon-like peptide-2 receptor activation in the rat intestinal mucosa.

Glucagon-like peptide-2 (GLP-2) increases small intestinal growth and function in rodents and human subjects. GLP-2 exerts its effects through a seven-transmembrane domain, G protein-coupled receptor (GLP-2R), stimulating cAMP generation and activating protein kinase A signaling in heterologous cell lines transfected with the GLP-2R. As intestinal cell lines expressing the GLP-2R have not been identified, we developed methods for studying GLP-2R signaling in the rat small intestinal mucosa in vitro. Isolated rat intestinal mucosal cells expressed mRNA transcripts for the GLP-2R, as well as for chromogranin A and beta-tubulin III, markers for enteroendocrine and neural cells, respectively. cAMP production in response to [Gly2]GLP-2, a degradation-resistant analog of GLP-2, was maximal at 10-11 m (268 +/- 93% of control, P < 0.001), with reduced cAMP accumulation observed at higher doses. The cAMP response was diminished by pretreatment with 10-9 m GLP-2, and was abolished by pretreatment with 10-6 m GLP-2 (P < 0.05), indicating receptor desensitization. GLP-2 treatment of isolated mucosal cells increased 3H-thymidine incorporation (to 128 +/- 8% of controls, P < 0.05), and this was prevented by inhibition of the protein kinase A pathway with H89. In contrast, GLP-2 did not affect p44/p42 MAPK phosphorylation or the levels of cytosolic calcium in the mucosal cell preparation. These results provide the first evidence that activation of the endogenous rat mucosal GLP-2 receptor is linked to activation of a cAMP/protein kinase A-dependent, growth-promoting pathway in vitro.