Antagonization of TNF attenuates systemic hemodynamic manifestations of envenomation in a rat model of Vipera aspis snakebite.

OBJECTIVES: Tumor necrosis factor (TNF) has been reported as a mediator of local tissue injury following snake envenomation in an intact rat model. We investigated whether systemic release of TNF occurs following Vipera aspis envenomation. We further analyzed the possible connection between envenomation-related hemodynamic depression and TNF antagonization (TNF antibodies or soluble TNF receptor). DESIGN: A prospective, randomized, controlled experimental study using a rat model for snake envenomation. SETTINGS: A medical university hospital research laboratory. INTERVENTION: Eighty rats (300-400 g) were divided into four groups (n = 20): control and three experimental groups. Intramuscular injection of V. asis 500 microg/kg was administered to the three experimental groups: venom only (group 1), venom and 40 microg anti-TNF antibodies (group 2), venom and 250 microg soluble TNF receptor (p55-R; group 3). Hemodynamic parameters were monitored up to 4 h following venom injection. MEASUREMENTS AND RESULTS: A significant hemodynamic deterioration (reduction in heart rate and blood pressure) occurred 30 min following venom injection in group 1 compared to groups 2 and 3, where hemodynamic parameters remained stable throughout the 4 h observation period. Serum levels of TNF were detected 15 min after venom injection and peaked after 2 h at 485+/-12 pg/ml. CONCLUSIONS: The hemodynamic consequences of intramuscular injection of V. aspis venom can be blunted in a rat by systemic antagonization of TNF activity prior to venom injection. The poisonous hemodynamic effects of the V. aspis venom might be caused by systemic release of TNF.

Soluble endothelial and platelet selectins in serum and ascitic fluid of women with ovarian hyperstimulation syndrome.

PROBLEM: The selectins, a group of cell adhesion molecules, are major mediators of inflammatory, immunologic, and angiogenic reactions. Their possible involvement and levels of the soluble isoform in serum and ascitic fluid of women with ovarian hyperstimulation syndrome (OHSS) were not previously evaluated. METHOD OF STUDY: This prospective, case-control study involved 16 women with OHSS. Ten matched women treated by controlled ovarian stimulation and eight healthy women with normal diagnostic laparoscopy served as controls. Serum and peritoneal fluid samples obtained from all subjects were assayed for soluble endothelial selectin (sES) and soluble platelet selectin (sPS) by specific enzyme-linked immunosorbent assay. RESULTS: Significantly higher levels of sES (median, 17.1 [9-41] vs 9.3 [2.4 21.3] ng/mL [P = 0.03]) and sPS (median, 23 [13-277] vs 6.5 [1.6-28.7] ng/mL [P = 0.001]) were observed in the peritoneal fluid of women with OHSS than the basal levels of healthy, non-stimulated women. Women with OHSS had significantly lower sES serum levels than those treated by controlled ovarian hyperstimulation without OHSS, while their sPS serum levels were comparable. CONCLUSIONS: Ascitic fluid of women with OHSS contains appreciable amounts of soluble selectins, suggesting their ovarian origin and possible involvement in the syndrome.

Serum levels of inhibin B, unlike inhibin A and activin A, are not altered in women with preeclampsia.

PROBLEM: To investigate whether inhibin A, inhibin B, and activin A serum levels are altered in women with preeclampsia. METHOD OF STUDY: Serum samples of 20 women with preeclampsia (study group) and 20 normotensive pregnant women, matched for maternal and gestational age and parity, were assayed for inhibin A, inhibin B and activin A by specific enzyme-linked immunosorbent assay. RESULTS: Median serum concentrations of inhibin A and activin A were significantly higher among women with preeclampsia than in women with normotensive pregnancies, while inhibin B levels were comparable in both groups. Activin A levels were positively correlated with those of inhibins A and B, and inhibin A levels were positively correlated with diastolic blood pressure and inhibin B concentration in the study group. CONCLUSIONS: Inhibin A and activin A, but not inhibin B, serum levels are markedly increased in women with preeclampsia. These hormones might serve as an endocrine marker for preeclampsia.

Do soluble cell adhesion molecules play a role in endometriosis?

PROBLEM: Endometriosis is a chronic inflammatory disease associated with diverse immunologic disturbances. Cell adhesion molecules are essential for the development of immune and inflammatory reactions. This study was conducted to investigate whether or not serum and peritoneal levels of soluble cell adhesion molecules are altered in women with endometriosis. METHOD OF STUDY: The study group comprised five women with moderate-to-severe endometriosis. Eight healthy women with a normal diagnostic laparoscopy served as controls. Serum and peritoneal fluid samples from both groups were analyzed for the soluble isoform of intercellular cell adhesion molecule-1 (sICAM-1). vascular cell adhesion molecule-1 (sVCAM-1), endothelial selectin (sES), and platelet selectin (sPS). RESULTS: Serum levels of sICAM-1 were significantly increased in women with endometriosis (median levels: 410.4 ng/mL; range: 233.9 ng/mL 598.4 ng/mL vs. 235.7 ng/mL; range: 187.4 ng/mL -323.7 ng/mL; P = 0.02). Although the levels of sVCAM-1, sES, and sPS in both samples were higher in the study group, the differences did not reach significance. CONCLUSIONS: Our results suggest a role of ICAM-1 in the pathophysiology of endometriosis. However. the role of other investigated cell adhesion molecules should be confirmed by further studies.

Vascular cell adhesion molecule-1 in normal, failed, and ectopic pregnancy.

PROBLEM: Vascular cell adhesion molecule-1 (VCAM-1) is involved in early pregnancy establishment. This study sought to determine whether soluble VCAM-1 (sVCAM-1) serum levels differ among normal, failed, and ectopic pregnancy, its capacity to serve as a marker for pregnancy viability or ectopic pregnancy, and its correlation with serum progesterone and beta human chorionic gonadotrophin (PHCG) levels. METHOD OF STUDY: Maternal serum samples were obtained from 20 women with ectopic, 10 with normal, and 10 with failed intra-uterine pregnancy, all of comparable gestational age. Samples were assayed for sVCAM-1, progesterone, and betaHCG by specific assays. RESULTS: The median serum level of sVCAM-1 was comparable between the three pregnancy types (normal: 578.3 ng/mL, range 434.4-699.5 ng/mL; failed: 567.8 ng/mL, range 401.9 669.5 ng/mL; and ectopic: 470.7ng/mL range, 328.2-1151.1 ng/mL). Serum levels sVCAM-1 were not significantly correlated with betaHCG or progesterone levels. CONCLUSION: sVCAM-1 is not appropriate to serve as a marker for pregnancy viability or ectopic pregnancy.

Xanthine oxidase and tumor necrosis factor alpha: possible mediators of remote tissue injury after viper envenomation.

BACKGROUND: Tumor necrosis factor is associated with various local and systemic inflammatory sequelae following snakebite. Xanthine oxidase is a principal mediator of remote tissue injury (e.g., lungs, heart, liver). OBJECTIVE: To investigate in a snakebite-like animal model the as yet unexplored role of TNF and XO in mediating organ damage following snakebite. METHODS: Sprague-Dawley rats were injected intramuscularly with a non-lethal 500 micrograms/kg dose of Vipera aspis venom (n = 10) or saline (n = 10). Blood pressure and heart rate were continuously monitored, TNF-alpha was measured in the blood, and total XO + xanthine dehydrogenase activity was assessed in various tissues. Lung histology and permeability indices were analyzed. RESULTS: Venom injection caused a significant (P < 0.05) reduction in both heart rate and invasive arterial pressure. The blood circulating TNF levels were significantly higher in the intoxicated group (P < 0.05 vs. saline group), with changes seen at 30 minutes from intoxication in both groups. Total XO + XDH activity in the kidney, lung and liver of the venom-injected group was significantly (P < 0.05) higher than in the saline group, while the activity in the heart was similar. CONCLUSIONS: The mediation of remote organ and hemodynamic changes following intramuscular injection of a non-lethal dose of Vipera aspis venom can be attributed partly to TNF and partly to XO. More research is needed to better understand the role of either compound and the time frame of their activity before specific antagonists can be introduced for snakebite management.

Levels of vascular endothelial growth factor are elevated in patients with ectopic pregnancy: is this a novel marker?

OBJECTIVE: To determine serum levels of vascular endothelial growth factor (VEGF) and evaluate their capacity to serve as a marker for the diagnosis of ectopic pregnancy (EP). DESIGN: Prospective, case-controlled study. SETTING: A tertiary care center. PATIENT(S): Twenty women with EP, 10 women with normal intrauterine pregnancy, and 10 women with abnormal intrauterine pregnancy, all at comparable stages of gestation. INTERVENTION(S): Serum samples were obtained from all women. MAIN OUTCOME MEASURE(S): All samples were analyzed for VEGF, progesterone, and beta-hCG by specific methods. RESULT(S): Women with EP had higher serum levels of VEGF than women with normal intrauterine pregnancy and women with abnormal intrauterine pregnancy (median levels, 226.8 pg/mL, 24.4 pg/mL, and 59.4 pg/mL, respectively). With a cutoff level of 200 pg/mL, serum VEGF could distinguish intrauterine from extrauterine pregnancy with a sensitivity of 60%, specificity of 90%, and positive predictive value of 86%. CONCLUSION(S): The increased serum VEGF levels in women with EP may facilitate this challenging diagnosis and reduce maternal morbidity and mortality.

Vascular endothelial growth factor is increased in patients with preeclampsia.

PROBLEM: This study was conducted to determine whether altered levels of vascular endothelial growth factor (VEGF) may play a role in the pathogenesis of preeclampsia. METHOD OF STUDY: Maternal plasma samples were collected from 19 patients with preeclampsia (group A) either before the onset of labor, or before induction of labor or medical intervention. Plasma samples were also obtained from 19 normotensive patients with uncomplicated pregnancies (group B), who were matched with the patients with preeclampsia for gestational age and parity. Samples were frozen at -70 degrees C until assayed for VEGF by a specific enzyme-linked immunoassay. RESULTS: The mean maternal age was similar in groups A and B. For both groups the VEGF was detectable in all plasma samples. However, the plasma concentrations of VEGF were significantly increased in the group A patients, compared with those in group B (median, 47 ng/ml; range, 10.6-72 ng/ml versus median, 13.6 ng/ml; range, 0.66-20 ng/ml; P < 0.001). In group A, a positive correlation was noted between VEGF concentrations and the systolic and diastolic blood pressure (r = 0.56; P = 0.01 and r = 0.48; P = 0.037, respectively). CONCLUSIONS: Maternal plasma VEGF levels were elevated in the patients with preeclampsia and correlated with the severity of hypertension, suggesting a role for VEGF in the pathogenesis of preeclampsia.

Analysis of Arg834Gln and Val902Glu type 2A von Willebrand disease mutations: studies with recombinant von Willebrand factor and correlation with patient characteristics.

Type 2A von Willebrand disease (vWD), the most common qualitative form of vWD, is characterized by a relative decrease in circulating intermediate and high molecular weight (HMW) multimers. We studied the biosynthesis of recombinant von Willebrand factor (vWF) containing each of two type 2A vWD mutations previously reported by us, Arg834Gln and Val902Glu. The structure of recombinant Arg834Gln vWF within transfected COS-7 cells and the secretion of HMW multimers were similar to wild type vWF. The normal transport and secretion of Arg834Gln vWF, categorizes it as a group II type 2A mutation. In contrast, the Val90-2Glu mutation resulted in intracellular proteolysis of vWF with the generation of a 176-kD fragment and retention of vWF between the endoplasmic reticulum and the Golgi complex. Moreover, the 176-kD fragment was also increased in plasma from patients with the Val902Glu mutation. Significantly impaired secretion and intracellular proteolysis of Val902Glu vWF categorizes a new sub-group of type 2A mutations. The intracellular proteolysis of vWF Val902Glu explains the lack of response to 1-deamino 8-D-arginine vasopressin (DDAVP) in patients who carry the mutation.

Identification of three candidate mutations causing type IIA von Willebrand disease using a rapid, nonradioactive, allele-specific hybridization method.

Type IIA von Willebrand disease (vWD), the most common type II vWD variant, is characterized by decreased binding of von Willebrand factor (vWF) to platelet glycoprotein Ib (Gplb) and by a decrease in large and intermediate vWF multimers. Mutations reported to cause vWD type IIA are clustered within the A2 domain of vWF, which is encoded by exon 28. Genomic DNA from affected members of 12 unrelated families with type IIA vWD were screened for these mutations by a rapid, nonradioactive, allele-specific oligonucleotide (ASO) hybridization method. Oligonucleotides containing each of eight mutations were cross-linked onto a nylon membrane by UV irradiation. A fragment of vWF exon 28 was amplified from peripheral blood leukocyte DNA using biotinylated primers and hybridized to the immobilized oligonucleotides. Positive signals were detected with an avidin-alkaline phosphatase conjugate and chemiluminescent substrate. Thus, in a single hybridization reaction, a patient sample could be analyzed for a large number of mutations simultaneously. Polymerase chain reaction (PCR) products from four patients did not contain any of the tested mutations and therefore were sequenced. Three additional candidate missense mutations, two of them novel, were identified: Arg(834)-->Gln in one patient, Gly(846)-->Arg in one patient, and Val(902)-->Glu in three ostensibly unrelated patients. By ASO hybridization, the mutations were confirmed in the affected patients and excluded in unaffected relatives and 50 normal controls. In one family, the Val(902)-->Glu mutation was shown to be a de novo mutation. This rapid screening method is applicable to other subtypes of vWD for which mutations have been identified.

The role of calcium in differentiation of leukemic cell lines.

Increased calcium influx associated with differentiation of four human myeloid leukemic cell lines: HL-60, KG-1, U-937 and K-562, to either monocytic or granulocytic direction was demonstrated. Calcium influx was measured employing two methods; measurement of radioactive calcium influx rate at 4 degrees C and employing the fluorescent probe, fura-2 acetoxymethyl ester. The increase in Ca2+ influx was demonstrated with three chemically unrelated differentiation inducers: retinoic acid, 1 alpha, 25 dihydroxy vitamin D3 and dimethyl sulfoxide. Inhibitors of calcium uptake such as verapamil diltiazem and cromolyn, partially reduced differentiation, suggesting that differentiation of myeloid leukemic cell lines is dependent on the availability of extracellular calcium.

Differentiation-associated changes of cation-transport activities in myeloid leukemic cell lines.

Induction of differentiation in HL-60 and U-937 leukemic cell lines, resulted in 1.5-10-fold increase in 45Ca2+ uptake. The increased 45Ca2+ uptake in the differentiating cells was inhibited by verapamil, cromolyn and amiloride. Elevation in Ca2+ uptake in differentiating cells was also demonstrated using the fluorescent probe, fura-2 acetoxymethyl ester. The increased 45Ca2+ uptake was accompanied by a decrease in ouabain-insensitive and -sensitive 86Rb+ uptake. Furthermore, correlation between the changes in these activities was observed. Modulation of extracellular pH affected differentiation: higher pH increased the extent of differentiation.

Phorbol-myristate-acetate-induced platelet aggregation in the presence of inhibitors.

4 beta-Phorbol-12-myristate-13-acetate (PMA) at 100 ng/ml was able to induce platelet aggregation in the presence of agents which inhibited aggregation, triggered by other agonists such as adenosine diphosphate sodium salt (ADP), thrombin and collagen. PMA induced aggregation in acid-citrate-dextrose platelet-rich plasma. 100 microM tetracaine, 5 microM bromophenacyl bromide and 0.2 mM mepacrine decreased PMA-induced aggregation by only 10% in contrast to their high inhibitory effect on other aggregation systems. However, 0.4 mM mepacrine did inhibit PMA-induced aggregation at the same rate as the other aggregation systems. 100 mg/ml vincristine slightly affected PMA-induced platelet aggregation, whereas cytochalasin B rather enhanced it. Nordihydroguaiaretic acid, 5, 8, 11, 14-eicosatetraynoic acid and p-nitrophenyl-phosphorylcholine had no effect on PMA- or collagen-induced platelet aggregation, partially inhibited aggregation triggered by ADP and strongly inhibited aggregation caused by thrombin. It is suggested that PMA exerts its effect on platelets mainly due to its ability to alter their membranes.