First-trimester prediction of gestational hypertension through the bioelectrical impedance analysis of the body composition.

OBJECTIVE: Obesity is a risk factor for the development of gestational hypertension, with important consequences for both the mother and fetus. This prospective observational study aims to propose an early prediction model of hypertensive disorders in pregnancy among obese women, through the bioelectrical impedance analysis (BIA) at the first trimester, thus allowing early recognition of obese women that are at risk to develop gestational hypertension, in order to target preventive interventions. PATIENTS AND METHODS: Singleton obese women (BMI ≥ 30 kg/m2) between the 9th and 12th week of pregnancy were included in the study. The exclusion criteria were chronic diseases, like type 2 diabetes mellitus, hypertension, and other medical pre-existing conditions. Eligible women were followed up at 20, 28, and 36 weeks of gestation by measuring blood pressure, weight, and body composition with the use of the BIA. The diagnosis of gestational hypertension was made after the 20th week of gestation. Pregnancy and perinatal outcomes were then recorded. RESULTS: Of the 479 women included in the study, 85 (17.7%) developed gestational hypertension; the remaining 394 (82.3%) resulted to be normotensive. A higher rate of nulliparous women was found in the hypertensive group (50.6% vs. 37.6%, p = 0.02), together with a higher rate of induction of labor (55.3% vs. 40.9%, p = 0.02) and of small for gestational age (SGA) newborns (12.9% vs. 6.9%, p = 0.03). Significant differences emerged in the body composition between the two groups already from the first trimester, indeed women developing gestational hypertension showed elevated values of Total body Mass, FM, FFM, TBW (p < 0.02), and of leg's FM, FFM (p < 0.006). At the multivariate logistics regression, the risk of developing gestational hypertension resulted higher in women with elevated total body water levels in the first trimester (OR 1.10 95% CI 1.04 -1.92). CONCLUSIONS: The BIA is a rapid, easy, non-invasive, and inexpensive tool to evaluate the body composition of obese pregnant women. It represents a promising predictor of hypertensive disorders in pregnancy, which allows an early identification of the patients at risk of developing gestational hypertension, thus opening a window of opportunity for strictly monitoring and target preventive intervention.

Molecular basis of complement resistance of human melanoma cells expressing the C3-cleaving membrane protease p65.

The molecular mechanism of complement resistance of the human SK-MEL-170 melanoma cell line was investigated. The cells have been shown to express the C3b-cleaving membrane protease p65. To delineate the molecular consequences of the C3b-cleaving activity for the complement cytotoxicity, the molecular events during the initiation (R24 monoclonal antibody, C1), amplification (C4, C3), and membrane attack (C5, C9) phases of complement were studied in comparison to a complement-susceptible human melanoma line (SK-MEL-93-2). No cleavage of C4b and C5b, 2 molecules structurally similar to C3b, was observed on the cells during classical pathway activation indicating the specificity of the p65 protease for the C3b molecule. The rapid degradation of C3b by p65 on the surface of complement-resistant SK-MEL-170 cells generates a M(r) 30,000 C3 alpha'-chain-fragment detectable as early as 1 min after complement activation, whereas no such fragment was present in detectable amounts on complement-susceptible cells. As a result of the rapid C3b proteolysis by p65 on resistant SK-MEL-170 cells, less C5 convertases are formed, which in turn results in the formation of a lower number of terminal complement components and membrane attack complexes. R24 antibody and C1q binding to the resistant cells was slightly lower as to susceptible cells. C4 binding studies, however, revealed that the observed difference in antibody and C1q binding has no influence on the complement resistance of SK-MEL-170 cells: significantly more C4b was bound to complement-resistant (1565 +/- 92 fg/cell) as compared to susceptible cells (715 +/- 31 fg/cell). On extraction of the molecular forms of C4 bound to the cell membranes, an additional high molecular weight C4 species--apparently a C4b-C4b homodimer--appeared only on the resistant SK-MEL-170 cells that may function as a residual back-up C5 convertase. Collectively, these results show that SK-MEL-170 human melanoma cells evade complement-mediated cytolysis despite sufficient activation of early components of the classical complement pathway by p65-mediated rapid degradation of surface-bound C3b, leading to a significant reduction in membrane attack complex formation. Thus, rapid cleavage of surface deposited C3b was established as a powerful mechanism of complement resistance.

Radiolocalization of human small cell lung cancer and antigen-positive normal tissues using monoclonal antibody LS2D617.

The murine monoclonal antibody LS2D617, which reacts with an antigen associated with human small cell lung carcinoma (SCLC), was tested in preclinical models to assess its potential for specific targeting of tumors in human SCLC cancer patients. LS2D617 detects a cell antigen on the surface of cultured SCLC and neuroblastoma cell lines. Scatchard analysis of the binding of LS2D617 to NCIH69 SCLC cells indicates an affinity constant of about 1 x 10(8) M-1 and an epitope expression level of approximately 2 x 10(6) antigenic sites/cell. Molecular weight analysis of the target antigen and antibody competition experiments showed that LS2D617 should be classified as a SCLC Cluster 1 antibody (i.e., reacts with the neural cell adhesion molecule). LS2D617 was labeled with 111In and tested for biodistribution (4, 24, 48, 72, and 96 h postinjection) in nude mice bearing the human SCLC NCIH69 tumor. Tumor values peaked at about 35% injected dose/g (Day 3) compared with about 8% injected dose/g for an irrelevant IgG1 antibody while normal tissue accumulation for both antibodies was about 2-8% injected dose/g. Immunohistochemical studies demonstrated that LS2D617 reacts with the central nervous system, peripheral nerves, endocrine tissues, and heart tissue of rabbits as it does in human tissues. The ability of LS2D617 to accumulate in vivo in normal tissues that express the specific target antigen was tested in rabbits. Rabbits given i.v. injections of 111In-LS2D617 or control labeled antibody were sacrificed at 48 h and tissues were examined by gamma well counting, autoradiography, and immunohistochemical staining for murine immunoglobulin. Specific uptake was seen in all sites defined as antigen positive by immunohistology (i.e., heart, liver bile duct, peripheral nerves, pituitary, adrenal), excepting the central nervous system (brain and spinal cord) which was inaccessible to antibody because of the blood brain barrier. The use of preclinical in vivo targeting models to assess tumor as well as antigen-positive normal tissue targeting should aid in the strategy of antibody-based therapeutic intervention of human cancer by providing insight into the potential for tumor targeting and normal tissue toxicity that may be encountered in the clinic.

Complement killing of human neuroblastoma cells: a cytotoxic monoclonal antibody and its F(ab')2-cobra venom factor conjugate are equally cytotoxic.

Only a few monoclonal antibodies mediate complement lysis of tumor cells, but for several antibodies it has been demonstrated that a complement-activating function can be introduced by covalent coupling of cobra venom factor (CVF), a non-toxic glycoprotein which is a structural and functional homologue of human complement component C3. In this study we compared the efficacy of complement killing of human neuroblastoma cells by the complement-activating monoclonal antibody 3F8 directed against the GD2 ganglioside antigen with that of its F(ab')2-CVF conjugate. At equal numbers bound per cell the 3F8 antibody and the 3F8 F(ab')2-CVF conjugate were found to be equally cytotoxic in the presence of complement from several species including human. Maximal killing reached up to 98%. The kinetics of killing and the bivalent metal requirement confirmed that the cytotoxic activity of the 3F8 antibody is mediated via the classical pathway and that of the 3F8 F(ab')2-CVF conjugate via the alternative pathway. To achieve a comparable degree of killing, an approximately eight-fold higher concentration of the 3F8 F(ab')2-CVF conjugate was required which appears to be a consequence of the approximately eight-fold lower binding activity of the 3F8 F(ab')2-CVF conjugate compared to the intact 3F8 antibody. Our data suggest that the coupling of CVF to non-cytotoxic antibodies allows the generation of conjugates with a cytotoxic activity similar to that of inherently cytotoxic antibodies.

Antibody conjugates with cobra venom factor. Synthesis and biochemical characterization.

Immunoconjugates are semi-synthetic hybrid proteins which bear great promise to become a new generation of anti-tumor agents. While many immunoconjugates have been shown to be selectively cytotoxic in in vitro model systems, dramatic in vivo anti-tumor effects have not been reported. To improve the activity of immunoconjugates, careful structure-function analyses have to be performed. We report here such an analysis for immunoconjugates consisting of a monoclonal anti-tumor antibody (MoAb) and cobra venom factor (CVF), the complement-activating glycoprotein from cobra venom, synthesized with the heterobifunctional crosslinking reagent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). It is shown that a reaction mixture after protein coupling contains free MoAb and CVF as well as hybrid proteins of different compositions (dimers (MoAb-CVF), trimers (MoAb2-CVF, MoAb-CVF2), tetramers (MoAb-CVF3, MoAb2-CVF2, MoAb3-CVF), and some higher oligomers). While free MoAb and CVF can be removed by size exclusion chromatography, separation of different oligomeric hybrid proteins is not possible by this method. From the biochemical characterization of the hybrid proteins, which included the determination of sedimentation coefficients, recording of circular dichroism spectra with subsequent determination of secondary structure, and ultrastructural analysis by transmission electron microscopy, it was concluded that the two proteins do not undergo major structural changes upon coupling, and that the coupling of the two proteins is random with no preferential relative orientation. The functional inactivation of CVF was substantial (approximately 70%) due to both derivatization with SPDP and subsequent conjugation to the MoAb, with conjugation being relatively more inactivating than derivatization. In contrast, the binding activity of the antibody was far less susceptible to inactivation. In conclusion, our data indicate that immunoconjugate synthesis with heterobifunctional crosslinking reagents results in a mixture of heterogeneous hybrid proteins and causes substantial functional inactivation. For successful in vivo anti-tumor activity of future immunoconjugates with CVF and other protein ligands better methods for immunoconjugate synthesis will have to be developed.

High-density miniaturized thermal shift assays as a general strategy for drug discovery.

More general and universally applicable drug discovery assay technologies are needed in order to keep pace with the recent advances in combinatorial chemistry and genomics-based target generation. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon in which substrates, inhibitors, cofactors, and even other proteins provide enhanced stability to proteins on binding. This phenomenon is based on the energetic coupling of the ligand-binding and protein-melting reactions. In an attempt to harness these biophysical properties for drug discovery, fully automated instrumentation was designed and implemented to perform miniaturized fluorescence-based thermal shift assays in a microplate format for the high throughput screening of compound libraries. Validation of this process and instrumentation was achieved by investigating ligand binding to more than 100 protein targets. The general applicability of the thermal shift screening strategy was found to be an important advantage because it circumvents the need to design and retool new assays with each new therapeutic target. Moreover, the miniaturized thermal shift assay methodology does not require any prior knowledge of a therapeutic target's function, making it ideally suited for the quantitative high throughput drug screening and evaluation of targets derived from genomics.

Additive cytotoxicity of different monoclonal antibody-cobra venom factor conjugates for human neuroblastoma cells.

Insufficient numbers of antigen molecules and heterogeneity of antigen expression on tumor cells are major factors limiting the immunotherapeutic potential of the few clinically useful monoclonal antibodies capable of mediating complement cytotoxicity and antibody-dependent cellular cytotoxicity. To overcome this limitation, we converted two non-cytotoxic monoclonal anti-neuroblastoma antibodies, designated 3E7 (IgG2b) and 8H9 (IgG1), and the non-cytotoxic F(ab')2 fragment of the cytotoxic monoclonal anti-GD2 antibody 3F8 (IgG3) into cytotoxic antibody conjugates by covalent attachment of cobra venom factor (CVF), a structural and functional homologue of the activated third component of complement. Competitive binding experiments confirmed the different specificities of the three antibodies. In the presence of human complement, all three antibody-CVF conjugates mediated selective complement-dependent lysis of human neuroblastoma cells. Consistent with the kinetics of the alternative pathway of complement, approximately seven hours incubation were required to reach maximum cytotoxicity of up to 25% for the 3E7-CVF conjugate, up to 60% for the 8H9-CVF conjugate, and up to 95% for the 3F8 F(ab')2-CVF conjugate. The different extent of maximal cytotoxic activity of the three conjugates was reflected by corresponding differences in the extent of binding of both unconjugated antibodies and the respective conjugates. Any combination of the three antibody-CVF conjugates caused an additive effect in complement-mediated lysis. Using a cocktail of all three conjugates, the extent of complement-mediated killing could be increased up to 100%. These data demonstrate that by coupling of CVF the relative large number of non-cytotoxic monoclonal anti-tumor antibodies of interesting specificity can be used to design cocktails of cytotoxic conjugates and, thereby, to overcome the problem of insufficient and heterogeneous antigen expression on tumor cells for immunotherapy.

Therapy of streptozotocin induced diabetes with a bifunctional antibody that delivers vinca alkaloids to IL-2 receptor positive cells.

IVA039.1 is a bifunctional antibody with specificity for the murine IL-2 receptor and vinca alkaloids. Biodistribution studies show that IVA039.1 can target and deliver vinca alkaloids to tissues that contain IL-2 receptor positive cells. Vinca alkaloids are lymphocytotoxic. Therapy of diabetic mice with IVA039.1 plus vincristine results in a significant decrease in the glucose levels of diabetic compared to untreated mice. The therapeutic effect of IVA039.1 plus vincristine therapy was additive but surprisingly not synergistic. The binding of IVA039.1 to vincristine has moderate affinity with a slow off rate. In vitro studies suggest that, when bound to IVA039.1, the vincristine is inactivated. We attribute the lack of an enhanced therapeutic response to bifunctional antibody therapy using IVA039.1 plus vincristine to the inaccessibility of the drug to the target cells.

Glucoregulatory endocrine responses to intermittent exercise of different intensities: plasma changes in a pancreatic beta-cell peptide, amylin.

Amylin, a peptide hormone released from the beta cells of the pancreas and cosecreted with insulin, is reported to inhibit the release of postprandial glucagon and insulin and to modulate gastric emptying. Changes in insulin and glucagon are important for controlling blood glucose levels under conditions in which metabolic rate is elevated, such as during and following exercise. Amylin may participate in the regulation of blood glucose levels in response to exercise, although the role of amylin has not been investigated. The purpose of the study was to determine the effects of a progressive, intermittent exercise protocol on amylin concentrations and to compare its response to circulating levels of insulin, glucagon, cortisol, and glucose. Seven well-trained males completed an intermittent exercise trial on a treadmill at four progressive exercise intensities: 60%, 75%, 90%, and 100% of maximum oxygen consumption (.VO(2)max). Blood samples were collected before exercise, after each exercise intensity, and for 1 hour following the exercise protocol. Subjects also completed a control trial with no exercise. Amylin and insulin rose from baseline (5.79 +/-.78 pmol/L and 4.76 +/-.88 microIU/mL) to peak after 100% .VO(2)max (9.16 +/- 1.35 pmol/L and 14.37 +/- microIU/ml), respectively and remained elevated during much of recovery. Thus, a progressive intermittent exercise protocol of moderate to maximum exercise intensities stimulates increases in amylin levels in well-trained individuals in a similar fashion to that of insulin, whereas glucagon concentrations only increase after the greatest exercise intensity, then quickly decline. Future studies should examine the effects of higher amylin concentrations in exercise recovery on glucoregulation.

Gestational weight gain in overweight and obese women enrolled in a healthy lifestyle and eating habits program.

OBJECTIVES: To determine whether changes in lifestyle in women with BMI > 25 could decrease gestational weight gain and unfavorable pregnancy outcomes. METHODS: Women with BMI > 25 were randomized at 1st trimester to no intervention or a Therapeutic Lifestyle Changes (TLC) Program including diet (overweight: 1700 kcal/day, obese: 1800 kcal/day) and mild physical activity (30 min/day, 3 times/week). At baseline and at the 36th week women filled-in a Food Frequency Questionnaire. OUTCOMES: gestational weight gain, gestational diabetes mellitus, gestational hypertension, preterm delivery. Data stratified by BMI categories. RESULTS: Socio-demographic features were similar between groups (TLC: 33 cases, CONTROLS: 28 cases). At term, gestational weight gain in obese women randomized to TLC group was lower (6.7 ± 4.3 kg) versus controls (10.1 ± 5.6 kg, p = 0.047). Gestational diabetes mellitus, gestational hypertension and preterm delivery were also significantly lower. TLC was an independent factor in preventing gestational weight gain, gestational diabetes mellitus, gestational hypertension. Significant changes in eating habits occurred in the TLC group, which increased the number of snacks, the intake of fruits-vegetables and decreased the consumption of sugar. CONCLUSIONS: A caloric restriction associated to changes in eating behavior and constant physical activity, is able to reduce gestational weight gain and related pregnancy complications in obese women.

Efficacy and tolerability of fitostimoline (vaginal cream, ovules, and vaginal washing) and of benzydamine hydrochloride (tantum rosa vaginal cream and vaginal washing) in the topical treatment of symptoms of bacterial vaginosis.

Two hundred and 91 patients showing signs and symptoms of bacterial vaginosis (BV) were randomized to receive topical treatment with Fitostimoline (vaginal cream and vaginal ovules + vaginal washing) or benzydamine hydrochloride (vaginal cream + vaginal washing) for 7 days. Signs (leucorrhoea, erythema, oedema, and erosion) and symptoms (burning, pain, itching, vaginal dryness, dyspareunia, and dysuria) (scored 0-3) were evaluated at baseline and at the end of treatment; the total symptoms score (TSS) was also calculated. In 125 patients, a bacterial vaginosis was confirmed by vaginal swab test. The primary efficacy variable analysis, that is, the percentage of patients with therapeutic success (almost complete disappearance of signs and symptoms), demonstrated that Fitostimoline ovules and vaginal cream were therapeutically equivalent and that pooled Fitostimoline treatment was not inferior to benzydamine hydrochloride. All the treatments were well tolerated, with only minor local adverse events infrequently reported. The results of this study confirmed that gynaecological Fitostimoline is a safe and effective topical treatment for BV.

Glycyl-tRNA synthetase of Escherichia coli: immunological homology with phenylalanyl-tRNA synthetase.

Antibodies to Escherichia coli glycyl-tRNA synthetase (GlyRS) cross-react extensively with E. coli phenylalanyl-tRNA synthetase (PheRS). These data indicate that structural homology exists between these two enzymes, the only two aminoacyl-tRNA synthetases in E. coli having an alpha 2 beta 2 subunit structure. Although only limited similarities are found in the protein sequences deduced from their known gene sequences, the presence of common epitopes in GlyRS and PheRS adds to a rather long list of physical and chemical similarities between those proteins. In addition, antibodies directed at the alpha- and beta-subunits of GlyRS inhibit both GlyRS and PheRS in the same relative manner, indicating that the function as well as the structure of subunits is similar in each enzyme. In contrast, GlyRS antibodies did not cross-react with a number of other aminoacyl-tRNA synthetase activities from E. coli, yeast, or Bacillus.

Acute colitis in the renal allograft recipient.

Four renal allograft recipients with evidence of ischemic damage to the colon are presented and compared with 11 cases from 5 major series. Similarities in the patients included: deterioration of renal function, multiple immunosuppressive and antibiotic regimens, the use of cadaver renal allografts, and diagnostic and therapeutic measures requiring frequent enemas with barium and ion-exchange resins. Two of our patients underwent surgery for the removal of segments of necrotic colon after several weeks of fever and abdominal pain initially attributed to either acute rejection, viral infection, or pancreatitis. One patient had three days of melena and responded to non-operative therapy. The fourth patient developed ischemic colonic changes 10 weeks after allograft nephrectomy and was receiving no immunosuppression at the time. Broad spectrum antibiotics were used at various times in all patients. Early aggressive evaluation of gastrointestinal complaints--including barium enema, upper gastrointestinal series with small bowel follow-through, proctosigmoidoscopy or colonoscopy, and arteriography--is indicated, in view of the lethality of the complication of colonic ulceration. The clinical pictures presented emphasize the fact that recipients of renal allografts are commonly heir to many complications which may be considered rare in the normal population.

A new end-to-end anastomosis formed without sutures for haemodialysis arteriovenous fistulas.

Carrying out vascular sutures is often a serious trouble during the surgical preparation of arteriovenous fistulas for haemodialysis. A new technique is suggested to perform an end-to-end anastomosis by inserting the artery 5-6 mm into the vein and then cementing the vessels by means of a cyanoacrilic tissue adhesive. The duration of the surgical procedure is reduced to half and this technique is much more simple than the usual ones. In 6 patients thus treated no troubles could be found either immediately or afterwards.

Immunoreactivity and function of oligosaccharides in cobra venom factor.

Cobra venom factor (CVF) is the nontoxic C-activating glycoprotein in cobra venom. It is a structural and functional analogue of complement component C3. Previous work has established that the major oligosaccharide chain of CVF is a symmetric fucosylated biantennary complex-type N-linked chain containing an alpha-galactosylated Le(x) antigenic epitope, Gal alpha 1-3Gal beta 1-4 (Fuc alpha 1-3) GlcNAc beta 1, a novel carbohydrate structure. We show that naturally occurring anti-alpha-Gal Ab present in normal human serum binds to CVF. In view of a possible human application of CVF, we studied the effect of this anti-alpha-Gal Ab on CVF function as well as the effect of oligosaccharide modification or removal on CVF activity and immunoreactivity with the anti-alpha-Gal Ab. The immunoreactivity of CVF with the anti-alpha-Gal Ab is abolished upon de-alpha-galactosylation or complete deglycosylation. De-alpha-galactosylated CVF and deglycosylated CVF were found to be equally active in forming a stable C3/C5 convertase with human factor B and in decomplementing human serum indicating that the oligosaccharide chains of CVF are not required for its C-activating function. Consistent with the unaltered functional activity, deglycosylation of the molecule caused only minor changes in secondary structure as judged by far UV circular dichroism analysis. However, the oligosaccharide chains appear to contribute to the thermal stability of CVF, because deglycosylation caused the molecule to be more sensitive to temperature. Inasmuch as rCVF produced in mammalian cells can be expected to contain sialylated oligosaccharide chains, we also prepared sialylated CVF by enzymatic sialylation of de-alpha-galactosylated and de-alpha-fucosylated CVF with 2,6-sialyltransferase. It was found that the secondary structure and the activity of sialylated CVF were unchanged compared to native CVF.