Adverse pregnancy outcome among teenagers: a reality?

The objective of this retrospective analysis was to evaluate maternal, fetal and neonatal outcomes in primi-adolescent pregnancies in Kuwait. Case records of primigravidae under 29 years of age, attending the antenatal clinic at our tertiary hospital, between January 2002 and December 2010, were analysed. The study group (up to 19 years of age at first pregnancy) consisted of 3,863 women and the control group (20-29 years of age at first pregnancy) comprised of 4,416 women. Maternal obstetric, fetal and neonatal complications were compared between the groups. Rates of ectopic pregnancy, pre-eclampsia, eclampsia, preterm labour, premature rupture of membrane and caesarean section were significantly higher among adolescents < 15 years of age; the risk then decreased steadily with age and became comparable with the control group after 16 years of age.

Absence of 5'-nucleotidase in porcine polymorphonuclear leucocyte membranes.

A fraction enriched in plasma membranes from porcine polymorphonuclear leucocytes, isolated by sucrose density centrifugation was shown to possess considerable AMP hydrolysing activity (150 nmol/min per mg protein). However all of this activity could be inhibited using excess p-nitrophenyl phosphate in the incubation medium. Furthermore the hydrolysis of AMP by the membrane was unaffected by the 5'-nucleotidase inhibitor alpha, beta-methyleneadenosine diphosphate and by the lectin concanavalin A, another potent inhibitor of 5'-nucleotidase. An antibody against mouse liver 5'-nucleotidase also did not inhibit the activity. These results suggest that the hydrolysis of AMP by porcine polymorph membranes is not accomplished by a specific 5'-nucleotidase and the necessity for distinguishing between true 5'-nucleotidase and non-specific phosphatase activity is discussed.

Activity of the glycosylating enzyme, core 2 GlcNAc (beta1,6) transferase, is higher in polymorphonuclear leukocytes from diabetic patients compared with age-matched control subjects: relevance to capillary occlusion in diabetic retinopathy.

The exact mechanism for capillary occlusion in diabetic retinopathy is still unclear, but increased leukocyte-endothelial cell adhesion has been implicated. We examined the possibility that posttranslational modification of surface O-glycans by increased activity of core 2 transferase (UDP-Glc:Galbeta1-3GalNAcalphaRbeta-N-acetylglucoaminyltr ansferase) is responsible for increased adhesion of leukocytes to vascular endothelium in diabetes. The mean activity of core 2 transferase in polymorphonuclear leukocytes isolated from type 1 and type 2 diabetic patients was higher compared with age-matched control subjects (1,638 +/- 91 [n = 42] vs. 249 +/- 35 pmol x h(-1) x mg(-1) protein [n = 24], P = 0.00013; 1,459 +/- 194 [n = 58] vs. 334 +/- 86 [n = 11], P = 0.01). As a group, diabetic patients with retinopathy had significantly higher mean activity of core 2 transferase compared with individuals with no retinopathy. There was a significant association between enzyme activity and severity of retinopathy in type 1 and type 2 diabetic patients. There was a strong correlation between activity of core 2 transferase and extent of leukocyte adhesion to cultured retinal capillary endothelial cells for diabetic patients but not for age-matched control subjects. Results from transfection experiments using human myelocytic cell line (U937) demonstrated a direct relationship between increased activity of core 2 transferase and increased binding to cultured endothelial cells. There was no relationship between activity of core 2 transferase and HbA(1c) (P = 0.8314), serum advanced glycation end product levels (P = 0.4159), age of the patient (P = 0.7896), and duration of diabetes (P = 0.3307). On the basis that branched O-glycans formed by the action of core 2 transferase participate in leukocyte adhesion, the present data suggest the involvement of this enzyme in increased leukocyte-endothelial cell adhesion and the pathogenesis of capillary occlusion in diabetic retinopathy.

The effect of aminoguanidine and tolrestat on glucose toxicity in bovine retinal capillary pericytes.

Cultured bovine retinal capillary pericytes (BRP) were used to investigate the effect of an aldose reductase inhibitor, tolrestat, and an inhibitor of advanced glycation end products (AGE) formation, aminoguanidine, on glucose toxicity. Glucose at high concentration reduced the replicative activity of pericytes in a dose-dependent manner. Tolrestat completely inhibited the production of sorbitol in cells exposed to a high concentration of glucose but failed to protect the cells from glucose toxicity. These results suggest that sorbitol accumulation in cells is probably not the major mechanism for glucose toxicity. In contrast, the addition of aminoguanidine at 10 mM concentration to the culture media protected pericytes from glucose toxicity. The degree of protected pericytes from glucose toxicity. The degree of protection was dose-dependent and evident at aminoguanidine concentration as low as 1 mM. The drug was only slightly toxic to BRP but induced morphological changes in pericytes with the loss of cellular processes and decreased cell spreading. This may suggest some action of aminoguanidine on the pericyte cytoskeleton. High concentration of glucose significantly increased the level of early glycation but not fluorescent AGE formation on BRP proteins. This was inhibited by the addition of aminoguanidine suggesting that glycation of cellular/membrane proteins and other mechanisms may play an important role in the toxic action of high glucose levels in cultured pericytes.

Radiosensitization of mammalian cells by transition metal complexes containing nitroimidazole ligands.

Various transition metal complexes containing nitroimidazoles as ligands have been shown to act as radiosensitizers of hypoxic cells in vitro. Sensitization by cis-Pt(II)Cl2 (nitroimidazole)2 complexes is no greater than that which can be demonstrated by the free nitroimidazole ligand alone. These results differ from those previously described for the compound FLAP where an ER of 2.4 was obtained at a concentration of 50 microM. We report that, even using the same treatment technique, the maximum ER that can be achieved is 1.2. Further, the sensitizing efficiency of FLAP cannot be improved when cells are kept in contact with the compound for 12 hours in air prior to deoxygenation and irradiation. In contrast, Rh(II) complexes show much greater sensitization than can be obtained with the free ligand and these compounds are in turn more efficient sensitizers than the comparable Pt(II) complexes.

Levels of vascular endothelial growth factor are elevated in the vitreous of patients with subretinal neovascularisation.

BACKGROUND: Vascular endothelial growth factor (VEGF) has been shown to play a major role in intraocular neovascularisation in ischaemic retinal diseases. Subretinal neovascularisation is an important cause of central visual loss, but little is known about the role of this growth factor in its pathogenesis. The aim of this study was to investigate the possible role of VEGF in the development of subretinal neovascularisation. METHODS: Undiluted vitreous samples were obtained from patients undergoing vitrectomy for removal of non-age-related subfoveal neovascular membranes (SFNM). For comparison vitreous from patients undergoing vitrectomy for idiopathic full thickness macular holes (FTMH) and proliferative diabetic retinopathy (PDR) was used. Indirect enzyme linked immunosorbent assay (ELISA), with an antibody directed against the conserved N-terminal region of human VEGF165, was used to determine vitreous levels of VEGF. The growth factor was also localised in the vitreous of patients with SFNM by western blot analysis. RESULTS: The mean (SE) VEGF concentration in the vitreous of patients with SFNM was 27.78 (2.22) ng/ml (n = 8), FTMH was 16.62 (0.9) ng/ml (n = 18), and PDR was 37.77 (3.28) ng/ml (n = 16). The differences between the PDR group and SFNM group versus the FTMH group were both significant (p = 0.0001 and p = 0.0015) as analysed by the Wilcoxon rank sum test). CONCLUSIONS: Vitreous levels of VEGF are significantly elevated in eyes with non-age-related subretinal neovascularisation compared with eyes with FTMH but not as elevated as in PDR. This suggests that VEGF is involved in subretinal angiogenesis.

Mechanism of cadmium resistance in cultured Indian muntjac fibroblasts.

The mechanism by which Indian muntjac fibroblasts (M cells) became resistant to the toxic effects of cadmium was investigated. The resistant lines, CCR5 and CCR20, were approximately 8-fold and 18-fold more resistant to acute cadmium exposures than the parental M cell line. Examination of the roles of metallothionein, glutathione and membrane permeability indicates that although resistance may be multifactorial, the major change involves decreased uptake of cadmium by resistant cells.

Pregnancy following infertility: a concern?

This study is to assess whether a waiting time to pregnancy (WTP) >1 year elevates the risk of adverse pregnancy outcomes in essentially healthy women with a single normal fetus at a gestational age of >20 weeks. Comparisons were made between those who conceived spontaneously with those who conceived after treatment for infertility. A retrospective analysis of 1,020 pregnancies occurring after a delay (WTP) of >1 year was undertaken over a 6-year period. Of these pregnancies, 24 resulted from advanced reproductive techniques and were excluded, as were four first trimester abortions, leaving 992 women. Of these 992, study group A (treated) consisted of 553 (56%) women, of whom 312 (56%) were primigravidae (A1). The pregnancy outcomes were compared with those of the remaining 439 (46%) women who conceived spontaneously, group B (control, untreated). Of these women, 234 (53%) were primigravidae (B1). Results suggested that primigravidae in the study group showed an increased risk of pre-term births and this risk was statistically significant between the gestations of 34 and 37 weeks. This risk remained elevated after adjustment for covariates. Pregnancies of women in group A1 were appreciably shorter than those of primigravidae who got pregnant without treatment (36.1 +/- 0.8 weeks vs 39.2 +/- 2.1 weeks); had lower birth weights (2684 +/- 481 g vs 3481 +/- 703 g) and were more likely to be admitted to the neonatal intensive care unit. Primigravidae among the treated group showed a statistically significant risk of caesarean births.

Clinical validation of a link between TNF-alpha and the glycosylation enzyme core 2 GlcNAc-T and the relationship of this link to diabetic retinopathy.

AIMS/HYPOTHESIS: Increasing evidence suggests that chronic, subclinical inflammation plays an important role in the pathogenesis of diabetic retinopathy. We recently reported that a glycosylating enzyme, core 2 beta-1,6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T), is implicated in increased leucocyte-endothelial cell adhesion in diabetic retinopathy via an upregulation mechanism controlled by TNF-alpha. SUBJECTS, MATERIALS AND METHODS: We examined the functional link between circulating TNF-alpha and the activity and phosphorylation of core 2 GlcNAc-T in polymorphonuclear leucocytes of patients with type 1 and type 2 diabetes. RESULTS: Plasma levels of TNF-alpha, although similar in patients with type 1 and type 2 diabetes, were significantly higher than in age-matched healthy controls, and correlated well with the severity of retinopathy. Core 2 GlcNAc-T activity followed the same trend and was associated with phosphorylation of the enzyme. Finally, the observation that TNF-alpha levels are also linked to glycaemic values suggests that in patients, as well as in vitro, the glycosylation-mediated cell adhesion process that plays a role in diabetic retinopathy may involve glucose- and TNF-alpha-induced protein kinase beta2 activation, and subsequently raise activity of core 2 GlcNAc-T through increased enzyme phosphorylation. CONCLUSIONS/INTERPRETATION: Our results reveal a novel rationale towards a specific treatment of diabetic retinopathy, based on the inhibition of core 2 GlcNAc-T activity and/or the blockage of cognate glycans.

Cadmium-induced multistep transformation of cultured Indian muntjac skin fibroblasts.

During the past five years we have made a series of cadmium-transformed and resistant fibroblast cell lines by continuous low-level exposure to cadmium. In the present paper we describe the use of four of these lines with varying degrees of transformation to investigate the multistep nature of cadmium carcinogenesis. These include: (a) M cell, an immortal but nontransformed muntjac skin fibroblast line; (b) CCR5, a morphologically transformed and cadmium-resistant line derived from M cells after 20-months continuous exposure to small step-wise increases in cadmium; (c) SCR5, a tumorigenic line derived by selection (in the absence of cadmium) of rapidly growing CCR5 agar colonies; (d) T1, a line derived from an SCR5 tumour growing in a nude mouse. We have compared the morphological characteristics of the four cell lines using light and electron microscopy and evaluated their ability to grow in liquid culture, soft agar and nude mice. We have also examined the changes which have occurred in their cytoskeletons and extracellular matrices using fluorescent antibodies to actin, tubulin and fibronectin and related these to the strength of their cell-cell and cell-substrate attachments and to their levels of transformation and tumorigenesis. We have shown that, while some changes occur in a single step (e.g. intracellular cytoskeletal changes), others are gradual (e.g. changes in extracellular matrix, focus formation and ability to grow in soft agar). We conclude that continuous exposure to low levels of cadmium can initiate growth and structural changes which subsequently lead to cell transformation and tumorigenesis on the removal of cadmium. Though change with cadmium was slow, many of the transformed characteristics are similar to those reported for viral and chemically transformed cells.

Subcellular fractionation of porcine neutrophils by nitrogen cavitation and sucrose-density-gradient centrifugation.

Neutrophils, isolated in large quantities from porcine blood were disrupted by nitrogen cavitation and separated by differential centrifugation into a nuclear fraction and a post-nuclear supernatant. The latter was subfractionated by sucrose density gradient centrifugation into cytosol, a fraction consisting of membrane vesicles and two granule-rich fractions. The membrane fraction accounted for 1.9% of the protein in the post-nuclear supernatant, the light granule fraction for 2.2% and the dense granule fraction for 4.2%. Catalase, lactate dehydrogenase and malate dehydrogenase were largely confined to the cytosol. The dense granule fraction contained the highest quantities of the hydrolytic enzymes, although the membrane fraction was also rich in alkaline and acid phosphatase and gamma-glutamyl transpeptidase activities. Electron microscopy of the membrane fraction showed intact membrane vesicles, whereas the granular fractions consisted of electron-dense, membrane-bound granules. Two granular fractions were isolated which contained granules of differing size and density. 3H-labeled wheat germ agglutinin bound to the surface of intact neutrophils and when these were disrupted and fractionated the membrane fraction showed a specific binding activity 16-times greater than that of the cavitated sample. The membrane fraction interacted with the detergent digitonin and as a result underwent density perturbation increasing from 1.13 g X cm-3 to 1.18 g X cm-3. Dodecylsulphate-polyacrylamide gel electrophoresis showed the membrane fraction to consist of at least 40 protein bands, with relative molecular masses ranging from 200 000-16 000. The granule fractions contained less protein bands, with a protein composition quite distinct from that of the membrane fraction.

Biochemical characterisation of porcine polymorphonuclear leucocytes: comparison with human polymorphonuclear leucocytes.

Polymorphonuclear leucocytes were isolated from pig blood relatively free from other cells and were characterised biochemically and morphologically and compared with human PMNLs. The activities of 16 enzymes of porcine and human PMNLs were measured and compared. Alkaline phosphatase, acid phosphatase, phosphodiesterase, gamma-glutamyl transpeptidase, NADH-cytochrome c oxidoreductase, malate dehydrogenase and acetylcholinesterase had higher specific activities in procine than in human cells. Alkaline phosphatase has an 87-fold higher specific activity in porcine than in human cells. beta-glucuronidase, lysozyme, beta-galactosidase, N-acetyl-glucosaminidase, beta-glucosidase, myeloperoxidase and catalase had higher specific activities in human than in porcine cells. beta-glucuronidase and myeloperoxidase showed over a 1000- and a 13-fold higher specific activity, respectively, in human than in porcine cells. Porcine PMNLs are readily available in large numbers and are recommended for studies of phagocytosis, chemotaxis and membrane biochemistry.

The mutagenic and carcinogenic properties of three second generation antitumour platinum compounds: a comparison with cisplatin.

The cytotoxicity, mutagenicity and transforming potentials of three second generation platinum compounds have been investigated in mammalian cells. All the compounds showed positive response in two assay systems in Chinese hamster V 79 cells, i.e. measurement of mutation induction at the HGPRT locus and of DNA damage as indicated by sister chromatid exchange frequencies. At equitoxic doses, the compounds in order of decreasing mutagenicities were cisplatin, spiroplatin, carboplatin and iproplatin. The BHK transformation assay reflected a similar order in the potential carcinogenicity of the drugs. Cisplatin was highly carcinogenic, followed by spiroplatin. In comparison, carboplatin and iproplatin were potentially weak carcinogens.

Cadmium induced changes in cell organelles: an ultrastructural study using cadmium sensitive and resistant muntjac fibroblast cell lines.

A detailed electron microscopy study of cadmium sensitive and resistant muntjac fibroblast cell lines has identified a wide range of intracellular damage following exposure to cadmium. Damaged organelles included cell membrane, mitochondria, Golgi cisternae and tubular network, chromatin, nucleoli, microfilaments and ribosomes. Although cell membrane damage was generally the earliest indication of adverse cadmium action, particularly with continuous cadmium exposures, cells could tolerate extensive membrane loss. Mitochondrial distortion and some damage to Golgi was also tolerated. The turning point at which cadmium became lethal was generally marked by a cascade of events which included damage to both nuclear and cytoplasmic components. These results for fibroblasts are discussed and compared with damage reported in other types of cells.

Properties of p-nitrophenyl phosphatase activity from porcine neutrophils.

p-nitrophenyl phosphatase activity is high in porcine neutrophils and was found in plasma membrane and granule fractions isolated from sucrose density gradients after nitrogen cavitation to disrupt the cells. Very little activity was found in the cytosol. The enzyme has optimum activity at alkaline pHs with a pH optimum of 10.3. The pH profile was fairly broad with activity still remaining at physiological pH. Orthovanadate was shown to be a potent competitive inhibitor of the enzyme with a Ki of 14 microM. Phosphate also inhibited but at millimolar concentrations and the two inhibitors bind in a mutually exclusive fashion. Evidence from experiments using divalent ion chelators and zinc ions suggested that the phosphatase is a zinc metalloenzyme. Beryllium was found to be a very potent, non-competitive inhibitor of the neutrophil enzyme (Ki = 1.1 microM). Levamisole and theophylline were both shown to be uncompetitive inhibitors of the porcine phosphatase (Ki = 0.2 mM and 1.2 mM respectively). The neutrophil phosphatase was inhibited by L-homoarginine but unaffected by L-phenylalanine and L-glutamate.

Intracellular protein glycation in cultured retinal capillary pericytes and endothelial cells exposed to high-glucose concentration.

There is now increasing evidence suggesting that non-enzymatic glycation (NEG) of proteins is involved in the pathogenesis of chronic diabetic complication. In this study we demonstrate that chronic exposure to high-glucose concentration leads to intracellular protein glycation in cultured bovine retinal capillary pericytes and endothelial cells. The level of intracellular protein glycation, as measured using a competitive enzyme-linked immunoabsorbant assay (ELISA), was found to increase in both pericytes and endothelial cells as function of time. As expected products of NEG were only detected when the Schiff base and the Amadori products were chemically reduced to glucitollysine by sodium borohydride. Despite the accumulation of early glycation products on cellular proteins there was no further rearrangement reaction into advanced glycation endproducts (AGEs), even after 12 days of incubation in high-glucose medium. Immunofluorescence microscopy demonstrated that the monoclonal antibody reacting with glucitollysine stains the cytoplasm of both pericytes and endothelial cells in a finely punctate pattern. Further studies using Western blot analysis suggested that a number of cellular proteins, including smooth muscle actin in pericytes, become rapidly glycated. The results from this in vitro study suggest that excessive accumulation of early products of non-enzymatic glycation in pericytes and endothelial cells may play an important role in the pathogenesis of diabetic retinopathy.

Changes in muntjac fibroblasts associated with the acquisition of cadmium resistance. A pre-resistance, transitional and post-resistance study.

A series of cell lines with different levels of resistance to continuous cadmium exposure has been developed from an immortal but non-transformed muntjac fibroblast cell line. Concentrations accepted in their culture medium range from 0.1 microM for the cadmium sensitive parent line to 5 microM for the intermediate "cadmium-tolerant" line, to 5, 10, 20 and 50 microM for the four "cadmium-resistant" lines. The present paper follows the morphological changes which accompanied the development of resistance through a 20-month pre-resistance period, a relatively abrupt 6-week transitional period and a 3-year post-resistance period, during which time levels of cadmium resistance were increased. Initial changes which led to the cadmium-tolerant CR5 cell line included (i) increased efficiency in autophagocytosing damaged cell components and in ridding the cell of residual waste materials, (ii) a reduction in fluid filled vacuoles and (iii) improved recycling and/or replacement of cadmium-damaged cell membrane. With the advent of cadmium resistance the intracellular damage necessitating these activities disappeared, yet the series of changes which occurred included a massive build-up of Golgi and the appearance of a trans-Golgi tubular network in addition to cytoskeletal and membrane changes. Though metallothionein levels are greater in the cadmium-resistant variants, their increase appears inadequate on their own to account for the high levels of resistance. The post-resistance changes which accompanied each step-up in cadmium resistance included further membrane and glycocalyx changes, in addition to continued increases in Golgi bodies and tubular network. This paper details the morphological changes which occurred throughout the 5-year period, tests the direct dependence of each on the presence of cadmium and examines their possible contribution to a cadmium protective mechanism.

Toxic action of advanced glycation end products on cultured retinal capillary pericytes and endothelial cells: relevance to diabetic retinopathy.

The toxic effects of advanced glycation end products (AGEs) on bovine retinal capillary pericytes (BRP) and endothelial cells (BREC) were studied. AGE-modified bovine serum albumin (AGE-BSA) was toxic to BRP. At a concentration of 500 micrograms/ml it reduced the BRP number to 48 +/- 3% (p < 0.05) of untreated controls, as determined by cell counting with haemocytometer. AGE-BSA was also toxic to bovine aortic endothelial cells (BAEC) reducing cell number to 84 +/- 3.1% of untreated controls. Under similar conditions, low concentrations (62.5 micrograms/ml) of AGE-BSA were mitogenic to BREC increasing the cell proliferation to 156 +/- 11% (p < 0.05) above that of untreated controls. At a higher dose of 500 micrograms/ml AGE-BSA decreased the proliferation of BREC to 85 +/- 6% of untreated controls. Immunoblot analysis demonstrated that BRP and BREC express the p60 AGE-receptor. Retinal capillary bed from the human also stained positively for the p60 AGE-receptor. Addition of 0.25 micrograms/ml of p60 AGE-receptor antibody was able to block the effects of AGE-BSA on BRP and BREC. The level of binding of [125I]-labelled AGE-BSA to the cell surface was small but significant among the three cell types. There was also an increase in the internalized pool of radioligand in BRP and BREC but this was very much lower than in BAEC. In all the cell types the internalized pool of [125I]-labelled AGE-BSA was much larger than the amount associated with the cell surface. Degradation products were not detected in the media over the 24-h incubation of the cells with [125I]AGE-BSA. The binding of [125I]-labelled AGE-BSA to the cell surface was prevented by the addition of p60 AGE-receptor. These results suggest that the interaction of AGE-modified proteins with the membrane-bound AGE-receptor may play an important role in the pathogenesis of diabetic retinopathy.

The interaction between radiation and complexes of cis-Pt(II) and Rh(II): studies at the molecular and cellular level.

A range of Rh(II) carboxylates and cis-Pt(II) complexes have been examined for their ability to increase the radiation sensitivity of aerobic and hypoxic V79 cells in vitro. The transition metal complexes sensitize in both air and nitrogen, with the greater effect generally occurring in nitrogen. The cis-Pt(II) complexes only show small levels of sensitization with dose modification factors (DMFs) of no more than 1.2. In contrast, the Rh(II) complexes can give DMFs of 2.0. Radiation chemical experiments show the transition metal complexes to have substantially lower redox potentials than metronidazole and, in addition, neither type of complex undergoes electron transfer reaction or adduct formation on interaction with radicals derived from DNA bases. Thus, the inorganic complexes do not operate by mechanisms similar to those occurring with electron affinic or stable free radical sensitizers. The increase in radiation sensitivity for cells treated with the Rh(II) carboxylates, but not the cis-Pt(II) complexes, is attributed to the ability of the Rh compounds to deplete intracellular thiols. Further, the efficiency of sensitization by the Rh(II) complexes and their ability to interact with cellular thiols depends upon the nature of the carboxylate ligand and follows the order butyrate greater than propionate greater than acetate greater than methoxyacetate. The differences between the carboxylates may be due to differences in drug uptake. A combination of the Rh(II) complexes with misonidazole given to hypoxic cells irradiated in vitro gives an additive response. However, it was not possible to demonstrate a similar effect in tumours in mice given the combination of Rh(II) methoxyacetate and the misonidazole analogue RSU 1070.