Andrés López Bernal

A novel method of purifying lung surfactant proteins A and D from the lung lavage of alveolar proteinosis patients and from pooled amniotic fluid

A simple procedure has been developed for the purification of the surfactant proteins SP-A and SP-D from lung lavage of patients with alveolar proteinosis. The SP-D is purified by fractionation of the supernatant obtained after spinning the lavage at 10000 X g for 40 min, while the bulk of the SP-A is purified by fractionation of the pellet. The supernatant is applied to a maltosyl-agarose column and the bound SP-D is specifically eluted using MnCl2. The pellet is solubilised in 6 M urea and, following renaturation, the solubilised proteins are applied to maltosyl-agarose and SP-A eluted using a gradient of EDTA. Both SP-A and SP-D are further purified by gel-filtration on Superose-6. This procedure has also been used to prepare successfully human SP-A and SP-D from amniotic fluid and may be generally applicable to the isolation of these surfactant proteins from lung washings obtained from other species.

Tumor necrosis factor-α selectively stimulates prostaglandin F2α production by macrophages in human term decidua

OBJECTIVE: Our objective was to investigate the effect of tumor necrosis factor-α on prostaglandin production by human term decidual cell subtypes in vitro. STUDY DESIGN: We measured the effect of tumor necrosis factor-α on prostaglandins F 2α , E 2 , D 2 , F metabolite, and E metabolite production by decidual cells ( n = 4) with radioimmunoassay. We used flow cytometry after labeling with an antibody to histocompatibility antigen DR, L243, which is specific for macrophages in this tissue, to prepare pure populations of decidual macrophages ( n = 3). Differences in prostaglandin output were analyzed by Wilcoxon and Kruskal-Wallis tests. RESULTS: Tumor necrosis factor-α stimulated prostaglandin F 2α , output by unfractionated decidual cells, without altering the output of any other prostaglandins. Tumor necrosis factor-α (10 nmol/L) increased prostaglandin F 2α production from decidual macrophages (HLA-DR-positive cells) by about threefold: from a median of 727 (range 423 to 1226) to a median of 1974 (range of 1550 to 2201), fmol/10 6 cells per 18 hours, as compared with a 1.4-fold increase from nonmacrophages from a median of 247 fmol/10 6 cells per 18 hours (range 125 to 611) to a median of 340 fmol/10 6 cells per 18 hours (range 201 to 505). CONCLUSION: Stimulation of decidual prostaglandin F 2α production by tumor necrosis factor-α may be important in the etiology of spontaneous labor at term or preterm labor associated with infection.

3 Biochemistry and physiology of preterm labour and delivery

Human parturition is associated with profound changes in uterine connective tissue affecting mainly the cervix, but the endocrine control of cervical ripening remains obscure. Connective tissue changes are also implicated in premature rupture of the membranes, a problem often associated with preterm delivery, and it is believed that local inflammatory infiltration may play a role in both this condition and cervical ripening, but it is difficult to define which changes precede parturition and which are a consequence of the trauma of labour. Chorioamnionitis can cause preterm labour by provoking the release of inflammatory mediators in the decidua/fetal membranes area and it is likely that activation of prostaglandin release by decidual macrophages is involved in triggering labour. However, the role of macrophages and other bone marrow derived cells in normal labour and in labour associated with chorioamnionitis needs to be defined. It is likely that treatment with a combination of antibiotics and prostaglandin synthase inhibitors and/or other anti-inflammatory drugs is the most appropriate therapeutic approach. Idiopathic preterm labour and spontaneous labour at term are probably due to changes in the sensitivity of the myometrium to endogenous agonists. Recent progress in cell signalling pathways, such as the characterization of regulatory G proteins and the cloning of hormone receptors, should clarify the mechanism of action of relaxing and contracting agents on myometrial cells and should provide the means for the development of new therapeutic agents of high effectiveness and selectivity. This approach should result in better management of both term and preterm labour.

EFFECT OF FENAMATES ON PROSTAGLANDIN E RECEPTOR BINDING

The effect of fenamates on prostaglandin E receptor binding was examined in myometrial samples collected at hysterectomy. Sodium meclofenamate and mefenamic acid inhibited binding of PGE2 to its specific receptor in a dose-dependent manner, with an ED50 of 20 mumol/l and 200 mumol/l, respectively.

Uterine Quiescence: The Role of Cyclic AMP

It is accepted that whilst hormones such as oxytocin, vasopressin and prostaglandin F2α induce myometrial contractions, essentially via an elevation of intracellular calcium, other ligands, such as β‐adrenoceptor agonists, calcitonin gene‐related peptide, and prostaglandin E2, promote uterine quiescence via their ability to increase intracellular cyclic AMP levels. At present, the exact factors initiating human parturition remain unknown, and labour may occur due to a loss of uterine quiescence, an increase in uterine contractility, or a combination of both. Whilst many studies have aimed to understand the mechanisms underlying uterine contractility there is a relative paucity of data regarding myometrial relaxation. We have verified the presence of mRNA encoding adenylyl cyclase (AC) isoforms I, II, III, V, VI, VII, VIII and IX in both non‐pregnant and pregnant human myometrium, and in isolated myometrial cells maintained in cell culture. Furthermore, by means of immunoblotting and immunocytochemistry, we have demonstrated the expression of these isoforms as membrane‐associated AC proteins, and identified changes in individual AC isoform expression during gestation. These findings illustrate the diversity of potential cAMP generating pathways in human myometrium, and the complexity of the signal transduction systems underlying uterine quiescence.

The presence of NK3 tachykinin receptors on rat uterus

The NK3 agonist, senktide, induced a potent contraction of rat uterus in the presence of tetrodotoxin, atropine and indomethacin, or the tachykinin receptor antagonists L-659877 and [D-Pro4,D-Trp7,9,10]substance P (4-11). Additional contractile and radioligand binding studies with receptor selective agonists and antagonists confirmed the presence of NK3 receptors and also revealed the presence of NK1 and NK2 receptors. The rat uterus is the second peripheral tissue in which a post-synaptic, non-neuronal NK3 receptor has been identified.

Ovarian response in three consecutive in vitro fertilization cycles.

OBJECTIVE This study was designed to assess the ovarian response in the same patient in consecutive IVF cycles. DESIGN Retrospective study. SETTING Assisted reproductive unit at a university hospital. PATIENT(S) One hundred ninety women who underwent three consecutive cycles of IVF. INTERVENTION(S) All women used a combination of pituitary desensitization and gonadotropin stimulation protocol and underwent oocyte retrieval. MAIN OUTCOME MEASURE(S) Number of follicles produced and number of oocytes retrieved. RESULT(S) There were no significant differences in the number of follicles produced, number of oocytes retrieved, and number of embryos created by the same woman among the three cycles of treatment. CONCLUSION(S) Consistent ovarian response can be achieved during the first three consecutive IVF cycles.

Thromboxane receptor signalling in human myometrial cells

We measured the effects of stable thromboxane A2 (TXA2) analogues on signalling in cultured human myometrial cells. U46619 and/or IBOP stimulated total inositol phosphates (IPs) and cAMP production, RhoA-associated protein kinase (ROK) activity and elevated intracellular calcium [Ca2+]i. Pretreatment of the cells with pertussis toxin did not inhibit IPs or [Ca2+]i production but the thromboxane receptor (TP) antagonist SQ-29548 did inhibit IPs and cAMP production, the elevation of [Ca2+]i, and the increase in ROK activity. Pretreatment with thapsigargin inhibited [Ca2+]i elevation. TP receptor-stimulated ROK activity was inhibited by the ROK inhibitor Y27632 while ROK activity was enhanced by the caspase 3 inhibitor, Z-DEVD-FMK. TP receptor-stimulated IPs production is additive to prostaglandin F2alpha (FP) or prostaglandin E (EP) receptor-stimulated IPs production and neither FP nor EP receptor-stimulated IPs production is inhibited by SQ29548. Thus cultured human myometrial cells express at least two functional TP receptor subtypes; TPalpha-like (cAMP-stimulating) and TPbeta-like (IPs, [Ca2+] and ROK-stimulating).

Myosin light chain kinase and the onset of labour in humans.

Myosin light chain kinase (MLCK) is essential for myometrial contractions induced by calcium‐mobilizing agonists. From the gene of vertebrate smooth muscle/non‐muscle MLCK there are at least four proteins expressed. We have found that both a > 200 and a 137 kDa MLCK are equally expressed in human non‐pregnant (NP) and term pregnant (P) uterine smooth muscle and confirmed that 19 kDa telokin (TK) is only expressed in P myometrium. In addition, we have observed that a MLCK immunogen at ∼ 60 kDa is only expressed in NP myometrium, suggesting that its expression is inhibited during normal pregnancy in a hormonally dependent manner. However, when we compared pregnant myometrium from patients delivered preterm (PT) (< 34 weeks gestation), but not in labour (NIL), with PT patients in labour (IL) we found that PT(IL) samples expressed the ∼ 60 kDa MLCK immunogen and thus displayed a NP phenotype whereas PT(NIL) samples did not express the protein and retained a pregnant phenotype. We hypothesize that the novel ∼ 60 kDa MLCK immunogen contributes to the aberrent contractility associated with preterm labour.

Fetal surfactant as a source of arachidonate in human amniotic fluid.

The factors responsible for the onset of labor in women are not well understood but it is clear that parturition is associated with increased production of prostanoids and release of arachidonic acid by intrauterine tissues. Pulmonary surfactant is secreted from the fetal lung into the amniotic fluid where its concentration increases toward term. In this paper we have shown that the ability of fetal surfactant to stimulate prostaglandin production by amnion cells is greatly enhanced by pre-incubating surfactant with amniotic fluid. This is due to the release of fatty acids, including arachidonate, from the lipids of fetal surfactant by the sequential action of phospholipase C and diglyceride lipase. Thus, in addition to providing the amnion with a source of arachidonate derived from the intracellular transfer of arachidonate from surfactant phosphatidylcholine to phosphatidylethanolamine and phosphatidylinositol in amnion cells, fetal surfactant also contributes to the pool of free arachidonate in amniotic fluid.