Status of p53 in first-trimester cytotrophoblastic cells.

p53 has been called the cellular gatekeeper of the genome because it can induce cell-cycle arrest in G1, apoptosis or affect DNA replication in response to DNA damage. As p53 has been observed in first-trimester cytotrophoblastic cells (CTB), but its expression in normal cells is generally not detectable because of its short half-life, p53 could play an important role in cellular differentiation and/or in the control of the invasion of trophoblastic cells; therefore, p53 status was investigated in these cells. Using different antibodies recognizing different epitopes of p53 protein, abundant p53 expression was observed both in nuclear and in cytoplasmic compartments of first-trimester CTB. Whereas p53 was detected in the nuclei of few trophoblastic cells with an antibody recognizing the N-terminal epitope of the protein, high expression level of p53 in the cytoplasm of CTB was detected with an antibody recognizing the middle part of p53. The lack of immunoreactivity of p53 with antibodies recognizing the epitopes located at the N-terminus of p53 and the high level of p53 protein observed in the cytoplasm of CTB suggest that the N-terminus of p53 is involved in the formation of complexes. These cytoplasmic complexes were detected under non-reducing conditions in western blot analysis and had apparent molecular weights (MW) of 195, 167 or 125 kDa. These complexes could prolong the half-life of p53 in the cytoplasm of CTBs. By contrast, in the nuclei of CTBs, p53 seems to be present as a tetramer.

Can we improve the diagnosis of rupture of membranes? The value of insulin-like growth factor binding protein-1.

Our objective was to assess the value of insulin-like growth factor binding protein-1 (IGFBP-1) and other tests for the diagnosis of rupture of the membranes (ROM). We included 49 women with suspected ROM. The gold standard for membranes status was defined based on clinical examination, ultrasonography, tests results (except IGFBP-1) and labour information. Sensitivity, specificity, positive predictive value and negative predictive value of each test were as follows, respectively: IGFBP-1 (86, 74, 73 and 87%); bromothymol (64, 100, 100 and 77%); fern test (62, 96, 93 and 75%) and ultrasonography (19, 100, 100 and 61%). The detection of IGFBP-1 in vaginal secretions has high sensitivity for the diagnosis of ROM.

Metalloproteinases and human placental invasiveness.

Matrix metalloproteinases (MMPs) are the main mediators of extracellular matrix degradation. This confers an important role to these enzymes in the invasion of the trophoblast cells where their expression are spatiotemporally regulated. The regulation of MMPs activity is complex and is established at different levels. Their expressions depend on various cis-elements in their gene promoter, and are induced or repressed by various soluble factors. After expression and secretion, the proMMPs must be activated through an activation network. Then, the enzyme activity is regulated by inhibition or stabilization. In this review, we shall focus on the expression, the role in invasion and the regulation of MMPs in the human placenta.

Displasia epifisaria de Meyer: un caso de diagnóstico tardío / Meyer epiphyseal dysplasia: a case of delayed diagnosis

Las displasias esqueléticas son un grupo de condiciones heterogéneas que hasta la fecha abarca cerca de 200 desordenes diferentes desde el punto de vista genético y clínico. Tienen como común denominador un trastorno del proceso normal de crecimiento y desarrollo del tejido óseo y sus precursores cartilaginosos. En este artículo se presenta el caso de una adolescente de 16 años con displasia epifisaria de Meyer cuyo diagnóstico fue tardío y que a diferencia de los reportados hasta la fecha requirió tratamiento quirúrgico debido a la sintomatología persistente, además se hace una breve revisión de la literatura contenida hasta la fecha en el Index.

Papel del ortopedista en el maltrato infantil: Descripción de tres casos y revisión de la literatura / Role of orthopedist in child abuse: report of three cases and literature review

El maltrato infantil es una práctica tan antigua como la humanidad misma. De acuerdo con la Organización Mundial de la Salud (OMS) es un problema de salud pública y cerca de 40’000.000 niños de edades entre 0-14 años son las víctimas. En Colombia las estadísticas reportan maltrato infantil en el 36% de los hogares. Las lesiones del sistema musculoesquelético y tejidos blandos son muchas veces la clave más evidente, así que debemos tener en cuenta algunos signos importantes a la hora de evaluar un niño con sospecha de este padecimiento. Los estudios han reportado que las fracturas son la segunda forma más frecuente de presentación de maltrato infantil. Esta información nos hace replantear estrategias encaminadas a prevenir y disminuir su incidencia de maltrato infantil. Como miembros de los organismos de salud, somos los primeros llamados a participar de manera activa y oportuna en la detección temprana de los casos de abuso infantil.

[Problems with feeding concentrated milk by-products to veal calves]. / Probleme bei der Verfütterung von konzentrierten Milchnebenprodukten an Mastkälber.

In Switzerland between 35,000 and 50,000 farm calves per year are fed rations containing concentrated whey. If the ration is balanced, whey has no adverse effects on health and growth rates of calves. Feeding whey to farm animals makes ecological and economical sense and constitutes a sound management for the disposal of milk by-products. The described case consisted of 53 calves of which 7 (13.2%) died within the feedlot-period. Based on clinical and management findings, salt-intoxication was diagnosed because of deprivation of free access to water. When large amounts of hypertonic feed containing low quality whey are fed to calves, their health is adversely affected. Therefore, article 16 of the Swiss Animal Protection Regulation should be changed.

Control of MMP-9 expression at the maternal-fetal interface.

Cytotrophoblastic cells (CTB) from first trimester placenta form columns of invasive CTB. This invasive behaviour is due to the ability of CTB to secrete matrix metalloproteinases (MMPs) since tissue inhibitor of MMP (TIMP) inhibits their invasiveness. Although CTB behave like metastatic cells, in vivo they are only transiently invasive (first trimester) and their invasion is normally limited only to the endometrium and to the proximal third of the myometrium. This temporal and spatial regulation of trophoblast invasion is believed to be mediated in an autocrine way by trophoblastic factors and in a paracrine way by uterine factors. Several types of regulators have been investigated: hormones, extra-cellular matrix glycoproteins and cytokines or growth factors. This review is not intended to be an exhaustive catalogue of potential regulators of trophoblastic MMP-9 secretion but is aimed at summarising the most important signalling pathways involved in MMP-9 regulation.

Biomechanical and biological considerations relating to the clinical use of the Point Contact-Fixator--evaluation of the device handling test in the treatment of diaphyseal fractures of the radius and/or ulna.

The Point Contact-Fixator (PC-Fix) was designed as part of the developmental evolution of more biological devices for internal fixations. It is a device characterized by minimal contact to the underlying periosteum and bone, hereby minimizing potential damage to perfusion. A multicentre handling test was conducted in 1993 and 1994 in six hospitals to assess its performance. A total of 83 fractured forearm bones (34 radii and 49 ulnae) in 52 patients were stabilized with a PC-Fix. The mean age of the patients was 37 years. There were 24 AO type A, 24 type B and 5 type C fractures. Nine fractures were open. Mean follow-up was 15.6 months; follow-up was 100%. All 21 surgeons involved considered the use of the PC-Fix self-explanatory and relatively easy. Handling difficulties were mainly related to insertion of the monocortical screws. In particular, the precision required for insertion, the inability to incline the screw and the inability to pull the plate to the bone using the screw were considered hard to deal with. Stripping of the hexagonal slot was a problem at removal of the implant, possibly related to overtightening of the screws at insertion. In 76 of 83 bones (49 of 53 forearms), the fractures united without additional surgery. The healing pattern typically showed early callus formation bridging the fracture, followed by progressive mineralization of the central radiolucent line. Complications included one infection, one late displacement and four delayed unions, all requiring revision surgery. In 8 forearms, plate removal was performed after a mean of 10.3 months. Two refractures occurred after removal of the implant 6 and 7 months postoperatively. In conclusion, the practical handling of the new implant was straightforward and easily learnt. The treatment failures observed were related to technical and strategic errors. We consider that, when using devices with a locking compression principle, a minimum number of three screws should be placed on each side of the fracture, that interfragmentary compression of simple fractures is desirable in order to increase stability and that, contrary to in vivo animal studies, early plate removal is not indicated. The trend in fracture fixation is to improve the biomechanical and biological properties of operating technique and the devices used. The PC-Fix has proven a useful step in the right direction in this evolutionary process.

Methodology of clinical trials focusing on the PC-Fix clinical trials.

Once development and mechanical and biological laboratory testing have been completed, new technologies to be used in orthopaedic and trauma surgery must be investigated in humans before they can be used routinely. Prospective clinical investigations with or without randomization to standard treatments conducted according to the current standards and guidelines for Good Clinical Practice must be performed to prove the safety and efficacy of the new device. Furthermore, these tests serve to determine the specific indications, contraindications, tips and tricks as well as the pitfalls and how to avoid them. Last, but not least, the study must result in improved teaching of the use of the device and in improved follow-up of patients. The basis of every conclusion drawn from such a study is the complete documentation of each single use of the new device. We present the modalities and methodology for conducting a prospective clinical multicentre investigation in trauma surgery, focusing on the clinical trials carried out on the Point Contact Fixator (PC-Fix), a device for the internal fixation of long bone fractures developed as part of the scientific evolution towards the Less Invasive Stabilization Systems (LISS) now being introduced into clinical practice. Four prospective multicentre clinical investigations with an overall number of 1,229 PC-Fixators implanted from October 1993 to May 1998 were performed. To our knowledge this is the largest prospective series ever reported in orthopaedic trauma surgery to test a new device before market introduction. Due to a special documentation and implant replacement procedure, every PC-Fix implantation was documented. Very few patients were lost to long-term follow-up due to the personal commitment of the study monitors. Regular personal visits of the study monitor to the investigating hospitals and close communication between the surgeons, the engineers responsible for development, the study monitor, and the study sponsor meant that problems were identified and dealt with professionally without compromising the health of the patient. Case report forms were concise and had to be transmitted without delay. Handling of radiographs has become easier thanks to the transmission of digital images to the study centre via the internet. There are some aspects specific to trauma surgery which require special consideration and planning is essential, but clinical trials can be performed effectively.

Biochemistry and molecular biology of trophoblast invasion.

Cytotrophoblastic cells (CTBs) from first-trimester placenta form columns of invasive CTBs. This invasive behavior is due to the ability of CTBs to secrete matrix metalloproteinases (MMPs), since tissue inhibitor of MMPs (TIMP) inhibits their invasiveness in the extracellular space. Although CTBs behave like metastic cells, in vivo they are only transiently invasive (first trimester), and their invasion is normally limited only to the endometrium and to the proximal third of the myometrium. This temporal and spatial regulation of trophoblast invasion is believed to be mediated in an autocrine way by trophoblastic factors and in a paracrine way by uterine factors. Several types of regulators have been investigated: hormones, extracellular matrix glycoproteins, and cytokines or growth factors. This review is not intended to be an exhaustive catalogue of potential regulators of trophoblast invasion but is aimed at summarizing the most important categories of factors affecting trophoblast-endometrium interactions.

Treating forearm fractures using an internal fixator: a prospective study.

Some major complications of internal fixation with plates, such as infections and disturbance of healing, have been shown to be related to necrosis of bone and to the soft tissues immediately deep to the plate. This is attributable to plate contact. To deal with this phenomenon, an internal fixator, the Point Contact Fixator, was developed according to a new concept. The Point Contact Fixator resembles a plate but functions like a fixator, that is, the fracture is stabilized using a splint fixed to the bone by monocortical, angularly locked screws that are designed not to exert pressure between the splint and the bone, thereby minimizing implant-to-bone contact. Vascular damage to the osseous blood supply consequently is avoided. The new internal fixator is the first of a new family of implants in addition to nails, plates, and external fixators. To study the potential of the Point Contact Fixator in a prospective study, 79 forearm fractures in 55 patients were treated in a consecutive series by one surgeon using the same technique throughout. Followup to union is reported for 100% of the patients. Handling the fixator was simple; healing was uneventful; and the rate of complication was low.

Leptin modulates extracellular matrix molecules and metalloproteinases: possible implications for trophoblast invasion.

Leptin is a circulating hormone which plays an important role in the regulation of energy balance, haemopoiesis and reproduction. Leptin and its receptor (leptin-R) are localized in human placental tissue but their function is not known. In this study we have investigated the expression of leptin and leptin-R in the human placenta with particular attention to extravillous cytotrophoblastic cell islands and cell columns which play a pivotal role in trophoblast invasion and placental growth. We demonstrate that leptin-R immunoreactivity shows a strong expression in the distal extravillous cytotrophoblastic cells of cell columns invading the basal plate, whereas leptin expression is homogeneously expressed in all the cellular components of cell columns. Since the invasive ability of the distally located extravillous cytotrophoblast of cell columns is known to be regulated by a variety of proteases and some extracellular matrix molecules, we tested the influence of leptin on the in-vitro production of matrix metalloproteinase (MMP)-2, MMP-9 and fetal fibronectin (fFN) by cytotrophoblastic cells. We demonstrate that leptin increases, in a dose-dependent manner, the secretion of immunoreactive MMP-2 and fFN and enhances the activity of MMP-9 in cultured cytotrophoblastic cells. Our results suggest that leptin and leptin-R could have a role in the invasive processes of the extravillous cytotrophoblastic cells by modulating the expression of MMPs. In addition, these results provide a foundation for studying pathological conditions characterized by insufficient or excessive trophoblast invasion.

Mechanisms of endometrial control of trophoblast invasion.

Tumour invasion and trophoblastic invasion share the same biochemical mediators: the matrix metalloproteinases (MMPs) and their inhibitors. MMPs are a family of enzymes capable of digesting the extracellular matrices of the host tissues. Human cytotrophoblastic cells are constitutively invasive and produce MMPs. Tissue inhibitors of metalloproteinases inhibit cytotrophoblastic invasion in vitro, indicating that MMPs are causally related to trophoblast invasion in the endometrium. In contrast to tumour invasion of a host tissue, trophoblastic invasion during implantation and placentation is controlled stringently in both space and time. The factors responsible for these important regulatory processes are unknown but studies in vitro indicate that endometrial cytokines and growth factors are possible candidates. Insulin-like growth factor binding protein 1, the major secretory product of the decidua, interleukin 1, interleukin 6, leptin and tumour necrosis factor alpha, all of endometrial origin, are stimulators of MMPs, whereas transforming growth factor beta inhibits the proteolytic activity of cytotrophoblastic cells. Unfortunately, the ways in which these individual factors interact to regulate trophoblast invasion are far from being understood.

Paracrine and autocrine regulators of trophoblast invasion--a review.

Cytotrophoblastic cells (CTBs) from first trimester placenta follow one of two existing differentiation pathways: villous CTBs (vCTBs) form a monolayer of polarized epithelial stem cells which proliferate and eventually differentiate by fusion to form a syncytiotrophoblast (STB) covering the entire surface of the villus, or they can break through the STB at selected sites (in anchoring villi) to form multilayered columns of non-polarized but invasive CTBs. In vitro, CTBs invade a reconstituted basement membrane, they thus behave like metastatic cells. This invasive behaviour is due to the ability of CTBs to secrete matrix metalloproteinases (MMPs) since tissue inhibitor of MMP (TIMP) inhibits their invasiveness. MMPs are a family of at least 17 human zinc-dependent endopeptidases collectively capable of degrading essentially all components of the extracellular matrix (ECM). Although CTBs behave like metastatic cells, in vivo they are only transiently invasive (first trimester) and their invasion is normally limited only to the endometrium and to the proximal third of the myometrium. This temporal and spatial regulation of trophoblast invasion is believed to be mediated in an autocrine way by trophoblastic factors and in a paracrine way by uterine factors. Several types of regulators have been investigated: hormones, cytokines, growth factors and ECM glycoproteins. This review is not intended to be an exhaustive catalogue of all the potential regulators but is aimed at emphasizing those factors relevant in trophoblast-endometrial interactions.

Effects of interleukin-6 (IL-6) on cytotrophoblastic cells.

Tumour invasion and trophoblastic invasion share the same biochemical mediators: the matrix metalloproteinases (MMP) and their inhibitors. In contrast to tumour invasion of a host tissue, trophoblastic invasion during implantation and placentation is stringently controlled both in tissue localization and developmental stage. The factors responsible for these important regulatory processes are unknown, but in-vitro studies point to endometrial cytokines and growth factors as possible candidates. Here we examined the possibility that interleukin-6 (IL-6), a trophoblastic and endometrial cytokine, represents such a regulatory factor. Purified first trimester cytotrophoblastic cells (CTB) were cultured for 4 days in presence or absence of increasing concentrations of IL-6. MMP-2 and MMP-9 bioactivity (zymography) and immunoactivity were measured in the culture supernatants together with total human chorionic gonadotrophin (HCG), fetal fibronectin (FFN) and leptin. IL-6 did not change the cytotrophoblastic secretion of FFN or total HCG. In contrast, this cytokine induced a dose-dependent stimulation of the leptin secretion and increased the activity, but not the immunoreactivity, of MMP-9 and MMP-2. These results indicate that IL-6 could be considered as an endometrio-trophoblastic regulator of cytotrophoblastic gelatinases.

Production and characterization of monoclonal antibodies against pregnancy-associated plasma protein A.

Pregnancy-associated plasma protein A (PAPP-A) was found to be a good first trimester maternal serum marker, together with free beta-human chorionic gonadotrophin (HCG) subunits, for the biochemical screening of fetal trisomy 21 (Down's syndrome). We have raised monoclonal antibodies (mAbs) against PAPP-A purified from human pregnancy serum. The different antibodies were characterized biochemically by Western blot analysis and in terms of specificity (reaction with non-pregnant and male serum). Their performance in Down's syndrome screening was assessed in comparison with an existing enzyme-linked immunosorbent assay method after labelling of the different mAbs with biotin or horseradish peroxidase. A pair of mAbs was eventually chosen for a double-antibody sandwich protocol. The selected combination was found to have a significantly increased specificity (P = 0.0116) over the method using (purified) polyclonal antibodies, together with slightly increased sensitivity. In our limited number of Down's syndrome pregnancy samples (n = 17) and controls (n = 18), the medians as well as the multiples of the median values (for the affected cases) were comparable between the two methods described.

Effects of tumour necrosis factor-alpha, interleukin-1 alpha, macrophage colony stimulating factor and transforming growth factor beta on trophoblastic matrix metalloproteinases.

The aim of this study was to determine the effects of tumour necrosis factor alpha (TNF), interleukin-1 alpha (IL-1alpha), macrophage colony-stimulating factor (MCSF) and transforming growth factor beta (TGFbeta) on the secretion of matrix metalloproteinases (MMP), human chorionic gonadotrophin (HCG) and fetal fibronectin (fFN) by purified first trimester cytotrophoblastic cells (CTB) in vitro. CTB were obtained from legal abortions and cultured in vitro in the presence or absence of the different cytokines. Secreted gelatinases were analysed in the culture supernatants by zymography, by measurements of the total gelatinolytic activity and by enzyme immunoassays. HCG and fFN were measured by commercially available immunoassays. TNF increased the total gelatinolytic activity by increasing MMP-9 activity (P = 0.025-0.0177) but decreased MMP-2 activity (P < 0.03) and immunoreactivity (P < 0.05), fFN (P < 0.02) and HCG (P < 0.01). IL-1alpha significantly increased the secretion of fFN (P < 0.02), the activity (P < 0.02) and immunoreactivity (P < 0.05) of MMP-9 but had no effect on the other parameters. MCSF increased MMP-9 immunoreactivity (P < 0.05) and moderately decreased HCG. TGFbeta inhibited total gelatinolytic activity, MMP-9 activity and immunoreactivity, but was without effect on MMP-2 concentrations and activity. TGFbeta decreased HCG (P < 0.041) and increased fFN (P < 0.042). Our results indicate that TGFbeta, TNF and IL-1alpha are important regulators of trophoblastic MMP secretion.

Involvement of trophoblast in embryo implantation: regulation by paracrine factors.

In order to investigate the regulatory role of only one endometrial cell type on trophoblastic invasion, we explored the effects of culture medium conditioned by in vitro decidualised stromal cells (DCM) and of insulin-like growth factor binding protein-1 (IGFBP-1, the main secretory product of decidual cells) on the trophoblastic secretion of gelatinases and tissue inhibitor of metalloproteinases (TIMP-1). First trimester cytotrophoblastic cells (CTB) were obtained from abortions and cultured in vitro in presence or absence of DCM or IGFBP-1. Secreted gelatinases were analysed in the culture supernatants by zymography and by measurements of the total gelatinolytic activity. Tissue inhibitor of metalloproteinases (TIMP-1) was measured by a commercially available immunoassay. DCM inhibited the total gelatinolytic activity of CTB but increased trophoblastic MMP-9 and TIMP-1. In contrast, IGFBP-1 increased the total gelatinolytic activity and TIMP-1 and had no effect on MMP-2 and MMP-9. We conclude that a factor secreted by decidual cells (possibly TGFbeta) inhibits the total gelatinolytic activity of trophoblast by increasing TIMP-1 but other factors, as yet unidentified, increase MMP-2 and MMP-9 to an extent which does not shift the equilibrium between the gelatinases and TIMP-1 in favour of the gelatinases. In contrast to DCM, IGFBP-1 increases the total gelatinolytic activity probably by stimulating another gelatinase (stromelysin-1?) as MMP-2 and MMP 9 are unchanged by IGFBP-1. The possibility of an integrin mediated effect of IGFBP-1 on CTB is discussed.

Effects of decidua-conditioned medium and insulin-like growth factor binding protein-1 on trophoblastic matrix metalloproteinases and their inhibitors.

The regulatory role of in vitro decidualized stromal cells (DESCM) and their main secretory product insulin-like growth factor binding protein-1 (IGFBP-1) was studied on the secretion of trophoblastic gelatinases and tissue inhibitor of metalloproteinase (TIMP-1). First trimester cytotrophoblastic cells (CTB) were obtained from abortions and cultured in vitro in presence or absence of DESCM or IGFBP-1. Secreted gelatinases were analysed in the culture supernatants by zymography and by measurements of the total gelatinolytic activity. TIMP-1, hCG, and fetal fibronectin (fFN) were measured by commercially available immunoassays. DESCM inhibited the total gelatinolytic activity of CTB but increased trophoblastic MMP-9, TIMP-1 and fFN. In contrast, IGFBP-1 increased the total gelatinolytic activity and TIMP-1, had no effect on MMP-2 , MMP-9 or fFN but inhibited hCG. It is concluded that a factor secreted by decidual cells inhibits the gelatinolytic property of trophoblast by increasing TIMP-1. Other decidual factors, as yet unidentified, increase MMP-2 and MMP-9 to an extent which does override the inhibitory effect of TIMP-1. Since in contrast to DESCM, IGFBP-1 increases the total gelatinolytic activity of CTB, it cannot be the primary active decidual factor regulating the proteolytic activity of CTB. The possibility of an integrin-mediated effect of IGFBP-1 on CTB is discussed.