Insulin-like growth factor I and risk of epithelial invasive ovarian cancer by tumour characteristics: results from the EPIC cohort.

BACKGROUND: Prospective studies on insulin-like growth factor I (IGF-I) and epithelial ovarian cancer (EOC) risk are inconclusive. Data suggest risk associations vary by tumour characteristics. METHODS: We conducted a nested case-control study in the European Prospective Investigation into Cancer and Nutrition (EPIC) to evaluate IGF-I concentrations and EOC risk by tumour characteristics (n=565 cases). Multivariable conditional logistic regression models were used to estimate associations. RESULTS: We observed no association between IGF-I and EOC overall or by tumour characteristics. CONCLUSIONS: In the largest prospective study to date was no association between IGF-I and EOC risk. Pre-diagnostic serum IGF-I concentrations may not influence EOC risk.

Anti-Mullerian hormone and risk of invasive serous ovarian cancer.

PURPOSE: Epithelial ovarian cancers either arise directly from Mullerian-type epithelium or acquire Mullerian characteristics in the course of neoplastic transformation. The anti-Mullerian hormone (AMH) causes regression of Mullerian structures during fetal development in males and has been shown to inhibit the growth of epithelial ovarian cancer. Therefore, we hypothesized that pre-diagnostic serum concentrations of AMH are inversely associated with risk of invasive serous ovarian cancer. METHODS: A case-control study (107 cases, 208 controls) was nested within the population-based Finnish Maternity Cohort (1986-2007). The sample donated during the first trimester of the last pregnancy preceding cancer diagnosis of the case subjects was selected for the study. For each case, two controls, matched on age and date at sampling, as well as parity at sampling and at cancer diagnosis were selected. AMH was measured by a second-generation AMH ELISA. Conditional logistic regression was used to compute odds ratios (OR) and 95 % confidence intervals (CI) for invasive serous ovarian cancer associated with AMH concentrations. RESULTS: Overall AMH concentrations were not associated with risk of invasive serous ovarian cancer (OR 0.93; 95 % CI 0.49-1.77 for top vs. bottom tertile, P trend=0.83). In women older than the median age at sampling (32.7 years), a doubling of AMH was associated with decreased risk (OR 0.69; 95 % CI 0.49-0.96), whereas an increased risk (OR 1.64; 95 % CI 1.06-2.54) was observed in younger women, P homogeneity = 0.002. CONCLUSIONS: In this first prospective investigation, risk of invasive serous ovarian cancer was not associated with pre-diagnostic AMH concentrations overall; however, the association may depend on age at AMH measurement.

The use of gravity or manual-stress radiographs in the assessment of supination-external rotation fractures of the ankle.

Supination-external rotation (SER) fractures of the ankle may present with a medial ligamentous injury that is not apparent on the initial radiographs. A cadaver gravity-stress view has been described, but the manual-stress view is considered to be the examination of choice for the diagnosis of medial injuries. We prospectively compared the efficacy of these two examinations. We undertook both examinations in 29 patients with SER fractures. Of these, 16 (55%) were stress-positive, i.e. and had widening of the medial clear space of > 4 mm with a mean medial clear space of 6.09 mm (4.4 to 8.1) on gravity-stress and 5.81 mm (4.0 to 8.2) on manual-stress examination, and 13 patients (45%) were stress-negative with a mean medial clear space of 3.91 mm (3.3 to 5.1) and 3.61 mm (2.6 to 4.5) on examination of gravity- and manual-stress respectively. The mean absolute visual analgoue scale score for discomfort in the examination of gravity stress was 3.45 (1 to 6) and in the manual-stress procedure 6.14 (3 to 10). We have shown that examination of gravity-stress is as reliable and perceived as more comfortable than that of manual stress. We recommend using it as the initial diagnostic screening examination for the detection of occult medial ligamentous injuries in SER fractures of the ankle.

Expression of androgen-binding protein (ABP) in human cardiac myocytes.

Cardiomyocytes are known to be androgen targets. Changing systemic steroid levels are thought to be linked to various cardiac ailments, including dilated cardiomyopathy (DCM). The mode of action of gonadal steroid hormones on the human heart is unknown to date. In the present study, we used high-resolution immunocytochemistry on semithin sections (1 microm thick), IN SITU hybridization, and mass spectrometry to investigate the expression of androgen-binding protein (ABP) in human myocardial biopsies taken from male patients with DCM. We observed distinct cytoplasmic ABP immunoreactivity in a fraction of the myocytes. IN SITU hybridization with synthetic oligonucleotide probes revealed specific hybridization signals in these cells. A portion of the ABP-positive cells contained immunostaining for androgen receptor. With SELDI TOF mass spectrometry of affinity purified tissue extracts of human myocardium, we confirmed the presence of a 50 kDa protein similar to ABP. Our observations provide evidence of an intrinsic expression of ABP in human heart. ABP may be secreted from myocytes in a paracrine manner perhaps to influence the bioavailabity of gonadal steroids in myocardium.

An alternate binding site for the P1-P3 group of a class of potent HIV-1 protease inhibitors as a result of concerted structural change in the 80s loop of the protease.

Structures of the complexes of HIV protease inhibitor L--756,423 with the HIV-1 wild-type protease and of the inhibitors Indinavir, L-739,622 and Saquinavir with the mutant protease (9X) containing nine point mutations (Leu10Val, Lys20Met, Leu24Ile, Ser37Asp, Met46Ile, Ile54Val, Leu63Pro, Ala71Val, Val82Thr) have been determined. Comparative analysis of these structures reveals an alternate binding pocket for the P1-P3 group of Indinavir and L--756, 423. The alternate binding pocket is a result of concerted structural change in the 80s loop (residues 79-82) of the protease. The 80s loop is pulled away from the active site in order to accommodate the P1-P3 group, which is sandwiched between the flap and the 80s loop. This structural change is observed for the complexes of the wild type as well as the 9X mutant protease. The study reveals that the 80s loop is an intrinsically flexible loop in the wild-type HIV-1 protease and that mutations in this loop are not necessary to result in conformational changes. Conformation of this loop in the complex depends primarily upon the nature of the bound inhibitor and may be influenced by mutations in the protease. The results underscore the need to understand the intrinsic structural plasticity of the protease for the design of effective inhibitors against the wild-type and drug-resistant enzyme forms. In addition, the alternate binding pocket for the P1-P3 group of Indinavir and L--756,423 may be exploited for the design of potent inhibitors.

Plasmid DNA vaccines: assay for integration into host genomic DNA.

The primary safety concern for DNA vaccines is their potential to integrate into host cellular DNA. We describe a sensitive and quantitative assay for investigating the tissue distribution and integration of plasmid DNA vaccines. By including gonadal tissues in the analysis, the potential for germline transmission is also assessed. At various time points after injection, total DNA is isolated from a variety of tissues and assayed by PCR for the presence of plasmid. To test for integration, genomic DNA is first purified away from free plasmid using a series of different gel electrophoresis procedures. The gel-purified genomic DNA is then assayed for integrated plasmid using PCR. Stringent methods are used to prevent contamination. The assay, validated using a variety of positive and negative controls, is capable of detecting one copy of plasmid per ug DNA (approximately 150,000 diploid cells). Using this assay, we have carried out intramuscular studies in mice or guinea pigs for four different DNA vaccine plasmids. There was no evidence of integration to a sensitivity of about one copy/microg DNA, which is at least three orders of magnitude below the spontaneous mutation frequency.

Non-active site changes elicit broad-based cross-resistance of the HIV-1 protease to inhibitors.

Three high level, cross-resistant variants of the HIV-1 protease have been analyzed for their ability to bind four protease inhibitors approved by the Food and Drug Administration (saquinavir, ritonavir, indinavir, and nelfinavir) as AIDS therapeutics. The loss in binding energy (DeltaDeltaG(b)) going from the wild-type enzyme to mutant enzymes ranges from 2.5 to 4.4 kcal/mol, 40-65% of which is attributed to amino acid substitutions away from the active site of the protease and not in direct contact with the inhibitor. The data suggest that non-active site changes are collectively a major contributor toward engendering resistance against the protease inhibitor and cannot be ignored when considering cross-resistance issues of drugs against the HIV-1 protease.

Therapeutic effects of nitric oxide inhibition during experimental fecal peritonitis: role of interleukin-10 and monocyte chemoattractant protein 1.

This study demonstrates that the therapeutic effect of a nitric oxide inhibitor in a murine model of fecal peritonitis is mediated in part by increased levels of interleukin-10 (IL-10) and monocyte chemoattractant protein 1 (MCP-1). Female CD1 mice were subjected to cecal ligation and puncture (CLP) with a 21-gauge needle and, immediately following surgery, were injected intraperitoneally with saline, N(G)-nitro-L-arginine methyl ester (L-NAME; 8 mg/kg), or N(G)-nitro-D-arginine methyl ester (D-NAME; 8 mg/kg). At 96 h after surgery and drug treatment, 20% of mice that received D-NAME had survived whereas 60% of mice that received L-NAME were alive. To elucidate the effect of L-NAME treatment on chemokine and cytokine production during fecal peritonitis, the levels of macrophage inflammatory protein 2 (MIP-2), IL-10, and MCP-1 were measured in peritoneal washings from additional groups of mice 24 h after the CLP surgery. Peritoneal fluids from L-NAME-treated mice contained significantly higher levels of IL-10 and MCP-1 than did those from D-NAME-treated mice. To elucidate the effect of nitric oxide inhibition on potential cellular sources of IL-10 and MCP-1 in the CLP model, cultured alveolar and peritoneal macrophages were activated with bacterial lipopolysaccharide in the presence of L-NAME; these macrophages produced significantly more MCP-1 than did similarly activated macrophages in the presence of D-NAME. In the CLP surgery model, immunoneutralization of IL-10 alone or IL-10 and MCP-1 together with polyclonal antibodies prior to surgery significantly reduced the survival rates in L-NAME-treated groups compared with L-NAME-treated groups that received preimmune serum. Taken together, these data demonstrate that the inhibition of nitric oxide following experimental CLP fecal peritonitis is therapeutic, in part through the modulatory effect of this treatment on the synthesis of IL-10 and MCP-1.

Mutational anatomy of an HIV-1 protease variant conferring cross-resistance to protease inhibitors in clinical trials. Compensatory modulations of binding and activity.

Site-specific substitutions of as few as four amino acids (M46I/L63P/V82T/I84V) of the human immunodeficiency virus type 1 (HIV-1) protease engenders cross-resistance to a panel of protease inhibitors that are either in clinical trials or have recently been approved for HIV therapy (Condra, J. H., Schleif, W. A., Blahy, O. M. , Gadryelski, L. J., Graham, D. J., Quintero, J. C., Rhodes, A., Robbins, H. L., Roth, E., Shivaprakash, M., Titus, D., Yang, T., Teppler, H., Squires, K. E., Deutsch, P. J., and Emini, E. A. (1995) Nature 374, 569-571). These four substitutions are among the prominent mutations found in primary HIV isolates obtained from patients undergoing therapy with several protease inhibitors. Two of these mutations (V82T/I84V) are located in, while the other two (M46I/L63P) are away from, the binding cleft of the enzyme. The functional role of these mutations has now been delineated in terms of their influence on the binding affinity and catalytic efficiency of the protease. We have found that the double substitutions of M46I and L63P do not affect binding but instead endow the enzyme with a catalytic efficiency significantly exceeding (110-360%) that of the wild-type enzyme. In contrast, the double substitutions of V82T and I84V are detrimental to the ability of the protease to bind and, thereby, to catalyze. When combined, the four amino acid replacements institute in the protease resistance against inhibitors and a significantly higher catalytic activity than one containing only mutations in its active site. The results suggest that in raising drug resistance, these four site-specific mutations of the protease are compensatory in function; those in the active site diminish equilibrium binding (by increasing Ki), and those away from the active site enhance catalysis (by increasing kcat/KM). This conclusion is further supported by energy estimates in that the Gibbs free energies of binding and catalysis for the quadruple mutant are quantitatively dictated by those of the double mutants.

Stem cell factor (c-kit ligand) influences eosinophil recruitment and histamine levels in allergic airway inflammation.

The increased reactivity of mast cells during allergic airway inflammation has been linked to several aspects of pulmonary disease. A primary inducer of mast cell differentiation, proliferation, and activation has been identified as c-kit ligand or stem cell factor (SCF). In the present study, we used an established murine model of allergic eosinophilic airway inflammation to examine the role of SCF during an Ag-specific airway response. Initial data demonstrates increased SCF protein production at 8 h postchallenge in both lungs and serum of allergen-challenged, but not vehicle-challenged, mice. The immunolocalization of SCF in Ag-challenged lungs suggested that macrophage populations were the primary source of SCF, while epithelial cell regions also stained positive. Intense immunohistochemical staining of macrophages in bronchoalveolar lavage samples recovered from Ag-sensitized mice indicate that these cells may be a significant source of SCF in the lungs. Alveolar macrophages from the airways of normal mice stimulated with either TNF (0.1-10 ng/ml) or IL-4 (10 ng/ml) produced significant levels of SCF. Furthermore, neutralization studies demonstrated that the inhibition of airway SCF during allergen challenge significantly decreased eosinophil, but not neutrophil, infiltration throughout the response. Furthermore, when mice were treated with anti-SCF Ab, histamine levels were significantly reduced at 8 h postchallenge, the time of significant SCF production. Together, these data indicate that the production of SCF during Ag-induced lung inflammation by alveolar macrophages can play a significant role in the subsequent recruitment of eosinophils, possibly via mast cell activation and degranulation.

Three-dimensional structure of a mutant HIV-1 protease displaying cross-resistance to all protease inhibitors in clinical trials.

Analysis of mutational effects in the human immunodeficiency virus type-1 (HIV-1) provirus has revealed that as few as four amino acid side-chain substitutions in the HIV-1 protease (M46I/L63P/V82T/I84V) suffice to yield viral variants cross-resistant to a panel of protease inhibitors either in or being considered for clinical trials (Condra, J. H., Schleif, W. A., Blahy, O. M., Gadryelski, L. J., Graham, D. J., Quintero, J. C., Rhodes, A., Robbins, H. L., Roth, E., Shivaprakash, M., Titus, D., Yang, T., Teppler, H., Squires, K. E., Deutsch, P. J., and Emini, E. A. (1995) Nature 374, 569-571). As an initial effort toward elucidation of the molecular mechanism of drug resistance in AIDS therapies, the three-dimensional structure of the HIV-1 protease mutant containing the four substitutions has been determined to 2.4-A resolution with an R factor of 17.1%. The structure of its complex with MK639, a protease inhibitor of the hydroxyaminopentane amide class of peptidomimetics currently in Phase III clinical trials, has been resolved at 2.0 A with an R factor of 17.0%. These structures are compared with those of the wild-type enzyme and its complex with MK639 (Chen, Z., Li, Y., Chen, E., Hall, D. L., Darke, P. L., Culberson, C., Shafer, J., and Kuo, L. C. (1994) J. Biol. Chem. 269, 26344-26348). There is no gross structural alteration of the protease due to the site-specific mutations. The C alpha tracings of the two native structures are identical with a root-mean-square deviation of 0.5 A, and the four substituted side chains are clearly revealed in the electron density map. In the MK639-bound form, the V82T substitution introduces an unfavorable hydrophilic moiety for binding in the active site and the I84V substitution creates a cavity (unoccupied by water) that should lead to a decrease in van der Waals contacts with the inhibitor. These changes are consistent with the observed 70-fold increase in the Ki value (approximately 2.5 kcal/mol) for MK639 as a result of the mutations in the HIV-1 protease. The role of the M46I and L63P substitutions in drug resistance is not obvious from the crystallographic data, but they induce conformational perturbations (0.9-1.1 A) in the flap domain of the native enzyme and may affect the stability and/or activity of the enzyme unrelated directly to binding.