α-Lipoic acid blocks the GMCSF induced protease/protease inhibitor spectrum associated with fetal membrane weakening in-vitro.

INTRODUCTION: We use an in-vitro human fetal membrane (FM) explant-based model to study inflammation-induced FM weakening, a prerequisite for PPROM. In this system, GMCSF is a critical intermediate, both necessary and sufficient for TNFα and thrombin induced FM weakening. α-Lipoic-acid (LA) blocks TNFα and thrombin, as well as GMCSF-induced weakening. Recently, we reported LA concomitantly blocks GMCSF-induction of MMPs 2, 9 and 10 and inhibition of TIMPs 1-3. The aim of this study was to show that LA blocks GMCSF-induced increases in additional proteases and reductions in additional protease inhibitors. METHODS: FM fragments were cultured±LA and then±GMCSF. In other experiments, weak versus strong, fresh FM were cultured without additions. Fragments were strength tested and media analyzed by multiplex protein ELISA for proteases and protease inhibitors. RESULTS: GMCSF induced FM weakening and concomitantly increased several Proteases (Cathepsin-S, Proteinase-3, Elastase-2) and decreased several protease inhibitors (NGAL, Cystatin-C, HE4 and Thrombospondin1). LA inhibited GMCSF-induced FM weakening and all enzymatic changes. Untreated weaker versus stronger regions of fresh FM showed comparable differences in proteases and protease inhibitor patterns to GMCSF-stimulated versus controls. CONCLUSION: LA blocks GMCSF-induced human FM weakening and associated protease increases and inhibitor decreases. The GMCSF-induced spectrum of protease/protease-inhibitor changes is similar to that in the natural weak FM fragments. In concert with previously reported GMCSF-induced changes in MMPs & TIMPs, these other protease and protease-inhibitor changes presumably facilitate FM weakening and rupture. LA blocks these GMCSF effects and therefore may be a useful agent to prevent PPROM.

CD200 is a useful diagnostic marker for identifying atypical chronic lymphocytic leukemia by flow cytometry.

INTRODUCTION: Immunophenotyping by flow cytometry is routinely employed in distinguishing between chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Inclusion of CD200 has been reported to contribute to more reliable differentiation between CLL and MCL. We investigated the value of CD200 in assessment of atypical CLL cases. METHODS: CD200 expression on mature B cell neoplasms was studied by eight-color flow cytometry in combination with a conventional panel of flow cytometry markers. The study included 70 control samples, 63 samples with CLL or atypical CLL phenotype, 6 MCL samples, and 40 samples of other mature B cell neoplasms. RESULTS: All CLL samples were positive for CD200, whereas MCL samples were dim or negative for CD200. Of the CLL samples, 7 were atypical by conventional flow cytometry, with Matutes scores ≤3. These cases were tested for evidence of a t(11;14) translocation, characteristic of MCL, and all were negative, consistent with their classification as atypical CLL. All these atypical CLL samples were strongly positive for CD200. CONCLUSION: CD200 proved to be a useful marker for differentiation between CLL and MCL by flow cytometry. In particular, CD200 was useful in distinguishing CLL samples with atypical immunophenotypes from MCL.

Decidual GM-CSF is a critical common intermediate necessary for thrombin and TNF induced in-vitro fetal membrane weakening.

INTRODUCTION: Inflammation/infection and decidual bleeding/abruption are highly associated with pPROM. As no animal model for pPROM exists, we have developed an in-vitro model system for the study of human fetal membrane (FM) weakening/rupture. Using it we have demonstrated that both TNF/IL-1 (modeling inflammation) and thrombin (modeling bleeding) weaken full thickness FM in a dose dependent manner concomitant with inducing biochemical changes similar to those seen in the FM physiological weak zone. METHODS: As the physiological site of infection and bleeding is the choriodecidua (CD), we modified our model system with full thickness FM tissue mounted on modified Transwell culture inserts to permit directional TNF/thrombin exposure on the decidua only (rather than both sides of the FM). After incubation, medium was sampled separately from the CD facing (maternal side) or from the amnion facing (fetal side) compartments and probed for cytokine release and confirmed with western blots. The FM was strength tested within the insert. RESULTS: Full-thickness FM fragments exposed to TNF or thrombin on CD side only showed dose dependent weakening and biochemical changes consistent with previous reports. Concomitantly, GM-CSF increased markedly on the CD but not the amnion side. Numerous proteases including MMP1 and MMP3 also increased on the CD side. Pre-incubation with GM-CSF antibody blocked both thrombin and TNF induced weakening. Finally, GM-CSF weakened FM in a dose dependent manner. DISCUSSION: GM-CSF is a critical common intermediate in the thrombin and TNF FM weakening pathways.

Haematopoietic SCT in severe autoimmune diseases: updated guidelines of the European Group for Blood and Marrow Transplantation.

In 1997, the first consensus guidelines for haematopoietic SCT (HSCT) in autoimmune diseases (ADs) were published, while an international coordinated clinical programme was launched. These guidelines provided broad principles for the field over the following decade and were accompanied by comprehensive data collection in the European Group for Blood and Marrow Transplantation (EBMT) AD Registry. Subsequently, retrospective analyses and prospective phase I/II studies generated evidence to support the feasibility, safety and efficacy of HSCT in several types of severe, treatment-resistant ADs, which became the basis for larger-scale phase II and III studies. In parallel, there has also been an era of immense progress in biological therapy in ADs. The aim of this document is to provide revised and updated guidelines for both the current application and future development of HSCT in ADs in relation to the benefits, risks and health economic considerations of other modern treatments. Patient safety considerations are central to guidance on patient selection and HSCT procedural aspects within appropriately experienced and Joint Accreditation Committee of International Society for Cellular Therapy and EBMT accredited centres. A need for prospective interventional and non-interventional studies, where feasible, along with systematic data reporting, in accordance with EBMT policies and procedures, is emphasized.

The effects of thrombin and cytokines upon the biomechanics and remodeling of isolated amnion membrane, in vitro.

Abruption-induced thrombin generation and inflammation/infection induced cytokine production have both been associated with fetal membrane (FM) weakening and preterm premature rupture of the fetal membranes (PPROM). Using our in vitro model system we have demonstrated that thrombin, and separately the cytokines, tumor necrosis factor-alpha (TNFα) and interleukin-1-beta (IL-1ß), remodel and weaken full thickness FM. Additionally, we have reported that the anti-oxidant and NFκB inhibitor, alpha-lipoic acid (LA), blocks these thrombin and cytokine induced effects. The purpose of these studies was to determine whether thrombin and cytokines directly weaken the amnion membrane (AM), the major load-bearing component of FM. Isolated AM or full thickness FM fragments from unlabored Cesarean deliveries were incubated with thrombin, TNFα, or IL-1ß, for 48 h. Rupture strength (breaking force) of each fragment was thereafter determined using our published methodology. Biochemical evidence of remodeling and apoptosis; immunoreactive Matrix Metalloproteinase 9 (MMP9), Tissue Inhibitor of Matrix Metalloproteinase 3 (TIMP3) and cleaved poly (ADP-ribose) polymerase (C-PARP) levels in tissue extracts, were determined by western blot and densitometry. Thrombin induced a dose-dependent weakening of isolated AM (P < 0.001) coupled with dose dependent increases in PARP cleavage, and reciprocal increases and decreases, respectively, in MMP9 and TIMP3 protein (all P < 0.01). Thrombin receptor activating peptide-6 (TRAP) also weakened isolated AM. Neither TNFα nor IL-1ß weakened isolated AM. However, both cytokines weakened AM when it was incubated together with the choriodecidua as part of full thickness FM (P < 0.001). Cytokine-conditioned choriodecidua medium also weakened isolated AM (P < 0.001). Under conditions in which cytokines weakened the AM, the changes in MMP9, TIMP3 and PARP cleavage were consistent with those seen after thrombin incubation. LA blocked the FM weakening and remodeling effects. In summary, thrombin weakens AM directly whereas cytokines weaken AM indirectly by causing the release of soluble intermediates from the choriodecidua.

Increased activity and improved outcome in unrelated donor haemopoietic cell transplants for acute myeloid leukaemia in Australia, 1992-2005.

BACKGROUND/AIM: Numbers of unrelated donor allogeneic haemopoietic cell transplants (HCT) for acute myeloid leukaemia have increased in Australia in recent years. The aims of this study were to investigate the components of this change and find contributing factors to changes in outcome. METHODS: The study method was a retrospective analysis of 213 consecutive first unrelated donor HCT for acute myeloid leukaemia performed within Australia for adult patients during the years of 1992-1997 (n= 43) and 1998-2005 (n= 170). RESULTS: The proportion of patients transplanted in first or second complete remission (CR) increased markedly from 21% in 1992-1997 to 52% in 1998-2005. The cumulative incidence of relapse at 1 year post HCT was significantly lower for the later cohort (22% vs 30%, P= 0.04) and for patients transplanted in CR compared with those not in CR (16% vs 31%, P= 0.01). The overall survival probability was significantly better at 5 years post HCT for patients transplanted in 1998-2005 compared with 1992-1997 (40% vs 21%, P= 0.04). Multivariate analysis identified five independent significant favourable factors for survival among the whole patient group: age under 40 years, transplant in CR1, CR2 or first relapse, patient CMV seronegativity, good performance status and year of transplant within 1998-2005. CONCLUSION: The later cohort of patients had improved survival even after allowing for the effects of age, remission status and other factors, which suggests a general improvement in the safety of the procedure over time, particularly for patients in early disease stages at transplant.

Alpha-lipoic acid inhibits thrombin-induced fetal membrane weakening in vitro.

Cytokine-mediated inflammation and abruption-induced thrombin generation are separately implicated in matrix metalloproteinase (MMP)-mediated weakening of fetal membranes (FM) leading to preterm premature rupture of the fetal membranes (PPROM). At term, FM of both labored vaginal and unlabored Cesarean deliveries exhibit a weak zone overlying the cervix exhibiting ECM remodeling characterized by increased MMP9 protein and activity. We have reproduced these biochemical changes as well as FM weakening in vitro using tumor necrosis factor-alpha (TNF) and interleukin (IL)-1ß, inflammatory cytokines implicated in PPROM. Additionally, we have reported that the antioxidant and NFκB inhibitor alpha-lipoic Acid (LA) blocks these TNF-induced effects. We now present the first direct evidence that thrombin also can induce FM weakening in vitro, and LA treatment inhibits this thrombin-induced-weakening. Full thickness FM fragments from unlabored Cesarean deliveries were incubated with increasing doses of thrombin (0-100 u/ml) for 48 h. Fragments were then strength tested (breaking force and work to rupture) using our published methodology. MMP3 and 9 levels in tissue extracts were determined by Western blot and densitometry. To determine the effect of LA, FM fragments were incubated with control medium or 10 u/ml thrombin, with or without 0.25 mM LA. Strength testing and MMP induction were determined. Thrombin induced a dose-dependent decrease in FM strength (42% baseline rupture force and 45% work to rupture) coupled with a dose-dependent increase in MMP3 and 9 expression (all p < 0.001). Treatment of FM with 0.25 mM LA completely inhibited thrombin-induced FM weakening and MMP expression (all p < 0.001). Thrombin treatment of cultured FM induces mechanical weakening and increased MMP3 and 9. Treatment of FM with LA inhibits these thrombin-induced effects. We speculate LA may prove clinically useful in prevention of PPROM associated with abruption.

Term human fetal membranes have a weak zone overlying the lower uterine pole and cervix before onset of labor.

The etiology of fetal membrane (FM) rupture is unknown. A hypothesis that the FM weakens by a process of collagen remodeling and apoptosis to facilitate rupture has been proposed. Human FMs reportedly exhibit a zone of altered histology, postulated to be the FM rupture site, but concomitant FM weakness has not been demonstrated. We hypothesized that a discrete zone of FM with marked weakness, histological change, and evidence of remodeling and apoptosis, develops in late gestation in the FM overlying the cervix. FM tissue from women undergoing prelabor cesarean delivery were perioperatively marked to identify the FM overlying the cervix, cut with a procedure that facilitates remapping the rupture strength of FM pieces to their former location and orientation on a three-dimensional model, and tested for strength. A 10-cm FM zone centered at the cervical mark was compared with the remaining FM. Mean rupture strength within the cervical zone was 55% of the remaining FM. The cervical zone also exhibited increased MMP-9 protein, decreased tissue inhibitor of metalloproteinases-3 (TIMP-3) protein, and increased PARP cleavage coincident with the previously reported zone of altered histology. A discrete zone of weakness is present in term prelabor FMs overlying the cervix and has biochemical characteristics consistent with tissue remodeling and apoptosis.

Stem cell transplantation for autoimmune disorders. Rheumatoid arthritis.

Haematopoietic stem cell transplantation (HSCT) is now being investigated as a potential therapy for patients with severe refractory rheumatoid arthritis unresponsive to conventional therapies, including tumour necrosis factor-alpha blockade. Pilot studies and an analysis of worldwide cases included in the European Group for Blood and Marrow Transplantation/Autologous Blood and Marrow Transplant Registry registry have enabled the progression of the technique. HSCT is well tolerated in patients with rheumatoid arthritis, and there has been no transplant-related mortality. Although HSCT is not a cure, an improved response to previously ineffective therapies is often seen. Further research is, however, required, and this procedure is still considered appropriate only for those patients for whom there is no reasonable therapeutic alternative. This paper reviews all the previous data relating to HSCT in rheumatoid arthritis, outlines the current stand and discusses future protocol options and research.

Vitamin C exacerbates hydrogen peroxide induced apoptosis and concomitant PGE2 release in amnion epithelial and mesenchymal cells, and in intact amnion.

This study was undertaken to investigate the effect of hydrogen peroxide (HP), a reactive oxygen species, and vitamin C, an antioxidant, on apoptosis and prostaglandin (PGE(2)) release in human amnion epithelial and mesenchymal cells, and intact amnion. Amnion cells and explants were incubated with and without HP and vitamin C. Cytoproliferation assay for viability, DNA fragmentation and PARP cleavage for apoptosis, EIA for PGE(2), and western blots for cyclooxygenases (COX) were performed. In amnion cells and explants, HP (0-5 mm) induced dose dependent apoptosis as per DNA fragmentation and PARP cleavage. HP (0-0.5 mm) also induced PGE(2)release concomitant with apoptosis in both cell types. In amnion explants, HP (0-10 mm) induced COX-2 protein and PGE(2)release concomitant with apoptosis. Vitamin C (0.01-10 mm), alone, enhanced epithelial but inhibited mesenchymal cell viability. It induced PGE(2)release in amnion explants. Vitamin C (1 mm) failed to inhibit HP induced apoptosis, but instead exacerbated it in epithelial and mesenchymal cells, and amnion explants. Vitamin C (0-10 mm) enhanced HP induced PGE(2)in mesenchymal cells. HP induces concomitant apoptosis and PGE(2)release in amnion epithelial and mesenchymal cells, and in intact amnion explants. HP induced apoptosis is not inhibited but enhanced by vitamin C.

Hematopoietic stem cell transplantation for severe rheumatoid arthritis.

The substantial morbidity and mortality associated with rheumatoid arthritis (RA), while not widely appreciated, provide adequate justification for consideration of high-dose immunoablative therapy followed by hematopoietic stem cell transplantation. While some patients with RA follow a benign course, selected subsets of patients have been identified with 5-year survival rates of 40-70%. A number of factors that can be easily determined serve as useful prognostic indicators for poor outcome. These include the presence of many involved joints (total joint count), the degree of functional disability as measured by the health assessment questionnaire and the presence of rheumatoid factor. This article summarises the present status of hematopoietic stem cell transplantation for rheumatoid arthritis and proposes future directions for research.

Physiological apoptotic agents have different effects upon human amnion epithelial and mesenchymal cells.

Foetal membrane rupture is thought to follow from gene-controlled tissue remodelling and apoptosis. We reported previously that staurosporine, cycloheximide, actinomycin D, as well as more physiological apoptotic agents (lactosylceramide, 15d-PGJ(2)) increase prostaglandin release in parallel with induction of apoptosis in WISH and amnion epithelial cells. Also, inhibition of prostaglandin release by cyclooxygenase inhibitors or PKA activators is accompanied by a parallel decrease in apoptosis. We hypothesize that amnion prostaglandin metabolism is linked with apoptosis in amnion epithelial cells and thus to membrane rupture. Amnion mesenchymal cells are also critical for membrane integrity. Their susceptibility to apoptotic agents is unknown and is the subject of this report. In amnion epithelial cells, lactosylceramide (125 microM) induced 6.5-fold, 20-fold increases in PGE(2) and NMP production (apoptosis), respectively. Conversely, in mesenchymal cells, lactosylceramide doses up to 200 microM had no effect on PGE(2) or NMP release. In both cell types, incubation with 15d-PGJ(2) (5-100 microM) demonstrated dose and time dependent increases in PGE(2) and NMP. PKA activators inhibited 15d-PGJ(2) induced PGE(2) release and apoptotis in epithelial cells, but not in mesenchymal cells, however. Major amnion cell types have different sensitivities to physiological apoptotic agents. Prostaglandin release occurs coincident with apoptosis in both amnion epithelial and mesenchymal cells.

Impact of race and gestational age on red blood cell indices in very low birth weight infants.

BACKGROUND: Normative data for hematologic values in the very low birth weight infants are limited and inconsistent, with the reported mean hematocrit (HCT) in these infants ranging from 43.5% to 60%. No data are available on the effect of race. OBJECTIVES: To establish normative data for hemoglobin (Hb) and HCT by arterial sampling obtained during the first 3 hours after birth in black and white premature infants

Effect of dexamethasone on lymphocyte subpopulations in premature infants with bronchopulmonary dysplasia.

OBJECTIVE: This study was designed to determine the effect of dexamethasone treatment on peripheral blood lymphocyte counts and subpopulations in premature infants with bronchopulmonary dysplasia (BPD). STUDY DESIGN: Peripheral blood lymphocyte subpopulations in 12 premature infants with BPD were analyzed before treatment with a 6-week course of dexamethasone (day 0), on days 3 and 10 of treatment, and 2 weeks after discontinuing dexamethasone therapy (day 56). Lymphocyte immunophenotypes were determined using direct two-color immunofluorescent staining followed by flow cytometry. RESULTS: The percentage of lymphocytes was significantly lower on days 3 (17.55 +/- 2.55) and 10 (20 +/- 11.8) of dexamethasone therapy compared with before (30.36 +/- 6.41) or after treatment. The percentage of T cells was significantly lower on days 3 and 10 of dexamethasone therapy (mean +/- SEM; 58.09 +/- 1.93 and 60.09 +/- 2.47, respectively) compared with before (67.09 +/- 4.24) or after treatment. The absolute number of T cells was significantly lower on day 10 of therapy. The percentage of CD4+ cells was significantly lower on days 3 (38.91 +/- 2.49) and 10 (40.45 +/- 2.24) of therapy, and this decrease persisted after dexamethasone was stopped (36.73 +/- 3.41). The absolute number of CD4 cells was significantly lower on day 10 (1328 +/- 216) of therapy and reached a nadir on day 56 (1143 +/- 106). Similarly, the CD4/CD8 ratio was also significantly lower on days 3 and 10 of treatment (1.56 +/- 0.18 and 1.64 +/- 0.14, respectively) and reached a nadir on day 56 (1.04 +/- 0.13). CONCLUSION: Dexamethasone significantly reduced the percentage and absolute number of lymphocytes, T cells, and CD4 cells, as well as the CD4/CD8 ratio. A reduction in CD4 cells and in the CD4/CD8 ratio persisted 2 weeks after dexamethasone therapy was stopped. In contrast, the absolute number of B cells increased transiently, and CD8 cells were unaffected by dexamethasone. This alteration in lymphocyte subpopulations may help account for the clinically beneficial anti-inflammatory effect of dexamethasone in the treatment of BPD complicated by respiratory failure. The dexamethasone-induced decrease in CD4 cells may also increase the susceptibility of these infants to infection.

Augmentation of antigen-specific lymphoproliferative responses in vitro by biological response modifiers.

The detection of antigen-specific T cell responsiveness, particularly of resting memory lymphocytes, in cultures of peripheral blood mononuclear cells (PBMC) may be hampered by a less than optimal antigen presentation in vitro. Augmented sensitivity of the test system may be achieved by the addition of reagents with a beneficial effect on lymphocyte and antigen-presenting cell (APC) functions. In this study the effect of several biological response modifiers on antigen-specific T cell proliferation was determined, using nickel sulphate and tetanus toxoid as test antigens. IL-1 alpha (100 U/ml), interferon-gamma (IFN-gamma) (10 U/ml), and indomethacin (2 microM) were found to significantly enhance nickel-induced proliferation in PBMC cultures from nickel-hypersensitive donors (n = 6). Tetanus-induced proliferation (n = 5) was similarly enhanced, both by the above supplements and by the addition of polyethylene glycol (PEG) or a neuraminidase treatment of the PBMC before culture. The addition to PBMC cultures of a combination of IL-1 alpha (30 U/ml), IFN-gamma (10 U/ml), and indomethacin (2 microM) is recommended to specifically enhance antigen-induced lymphoproliferative signals.

Protein kinase A activators inhibit agonist induced prostaglandin production in human amnion.

Prostaglandin (PG) production by human amnion has been postulated to have a role in the onset of labor. Previous work by ourselves and others has demonstrated that oxytocin, phorbol esters and epidermal growth factor (EGF) increase PGE2 production in human amnion cells by activation of the Phospholipase C/Protein Kinase C (PKC) cascade system. The present study was undertaken to determine the effect of prior activation of the Adenylate Cyclase cascade system upon subsequent stimulation of PGE2 production by oxytocin, phorbol 12-myristate-13-acetate (PMA) or EGF in amnion cells and membrane discs. Isoproterenol, forskolin and dibutyryl cyclic adenosine monophosphate (dbcAMP) were utilized to activate the Adenylate Cyclase system at the receptor, enzyme and second messenger level. In control amnion cells, oxytocin, PMA and EGF each provoked dose dependent increases in PGE2 production. In cells preincubated with dbcAMP, forskolin or isoproterenol, agonist stimulated PGE2 production was markedly (50-90%) inhibited (p < 0.01). Inhibition was dose dependent upon preincubator concentrations. Maximal inhibition by adenylate cyclase activators occurred with 2-4 h of preincubation. In membrane discs, forskolin preincubation also inhibited oxytocin, PMA and EGF stimulation of PGE2 production. Activation of the Adenylate Cyclase system in human amnion cells or membrane discs inhibits the subsequent action of potent stimulators of PGE2 production in human amnion.

Ritodrine: a beta-adrenergic receptor antagonist in human amnion.

OBJECTIVE: Human amnion is important in the initiation of labor. Ritodrine, when administered as a tocolytic, is found unchanged in amniotic fluid. We characterized effects of ritodrine binding to beta-adrenergic receptors in amnion and amniocytes. STUDY DESIGN: Iodine 125-iodopindolol, beta-adrenergic receptor agonists, and beta-adrenergic receptor antagonists were used to describe binding characteristics. Experiments were designed with and without isoproterenol and ritodrine to study intracellular cyclic adenosine 3'5'-monophosphate and prostaglandin E2 release. RESULTS: Scatchard analysis revealed a single class of saturable binding sites, with maximum binding capacity of 70.0 +/- 17.2 fmol/mg protein (n = 12) and with high-affinity dissociation constant of 458.9 +/- 72.1 pmol/L. Agonists and antagonists competed for the 125I-iodopindolol binding site consistent with a beta 2-adrenergic receptor. Hill coefficients were 0.6 to 0.8 for agonist competition and 1.0 for antagonist competition and ritodrine. Stimulation with isoproterenol resulted in dose-dependent increases in cyclic adenosine 3'5'-monophosphate and prostaglandin E2. Ritodrine failed to stimulate cyclic adenosine 3'5'-monophosphate and inhibited isoproterenol-stimulated cyclic adenosine 3'5'-monophosphate and prostaglandin E2 production. CONCLUSION: In human amnion binding of ritodrine to beta 2-adrenergic receptors and lack of ritodrine-mediated postreceptor effects are characteristic of a beta 2-adrenergic antagonist.

Human T lymphocyte cAMP-dependent protein kinase: subcellular distributions and activity ranges of type I and type II isozymes.

The role of the type I and type II protein kinase A isozymes in the regulation of human T lymphocyte immune effector functions has not been ascertained. To approach this question, we first characterized the distribution and enzyme activities of the type I and type II protein kinase A (PKA) isozymes in normal, human T lymphocytes. T cells possess both type I and type II isozymes with an activity ratio of 5.0:1 +/- 0.71 (mean +/- SD). The type I isozyme associates predominately with the plasma membrane whereas the type II isozyme localizes primarily to the cytosol. Analyses of isozyme activities demonstrated that T cells from approximately one-third of 16 healthy donors exhibited significantly higher type II isozyme activities (higher type II, type IIH) than the remaining donors (lower type II, type IIL) (mean = 605 +/- 75 pmol.min-1.mg protein-1, P less than 0.001). Scatchard analyses of [3H]cAMP binding in the cytosolic fraction demonstrated similar Kd values (type IIH, 1.1 x 10(-7) M; type IIL, 9.0 x 10(-8) M); however, the Bmax (maximal binding) of the type IIH was 400 fmol/mg protein compared to the Bmax of the type IIL of 126 fmol/mg protein. Scatchard analysis of [3H]cAMP binding to the type I isozyme associated with membrane fragments had a Kd of 5.6 x 10(-8) M and a Bmax of 283 fmol/mg protein. Eadie-Hofstee plots of type IIH and type IIL gave a Km and Vmax of 2.3 mg/ml and 1.5 nmol.mg-1.min-1, and 2.1 mg/ml and 1.6 nmol.mg-1.min-1, respectively. The 3.2-fold higher maximal binding of the type II isozyme in one-third of healthy donors may reflect a greater amount of isozyme protein. The compartmentalization of type I PKA isozyme to the plasma membrane and type II PKA isozyme to the cytosol may serve to localize the isozymes to their respective substrates in T lymphocytes.

The adenylate cyclase system and prostaglandin production in human decidua parietalis.

We studied the beta-adrenergic system in the human decidua and its effect on prostaglandin E2 and F2a release from dispersed decidual cells in culture. Beta-adrenergic receptors in decidual membranes were partially characterized using (+)[125I]HYP. Specific binding was demonstrated with maximum binding capacity (Bmax) of 70 +/- 10.6 fmol/mg and an affinity (KD) of 20.85 +/- 1.86 pmol. cAMP dependent phosphoproteins in decidual cytosol were also identified. Two phosphoproteins of M(r) 42,000 and 22,000 were seen in all preparations. Three others (M(r) 39,000, 23,000 and 21,000) were identified in only some of the preparations. Phosphoproteins of similar M(r) to those seen in cytosol prepared from decidual homogenates were also identified in cytosol of cultured decidual cells. Phosphorylation of the 42,000 M(r) and 22,000 M(r) proteins was maximal (3.04 +/- 0.35-fold and 5.7 +/- 0.68-fold) with 10(-6) M cAMP. Cultured decidual cells produced prostaglandin E2 and F2a which increased in a dose-dependent manner in response to dbcAMP, forskolin or isoproterenol. The decidua contains an intact beta-adrenergic system that, when activated, is capable of phosphorylating specific proteins and modulating prostaglandin release.