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PDF file - 121K, Knockdown of MMP-14 following MMP-14 RNAi of OVCAR-4 and OVCAR-3 cells

PDF file - 420K, MMP-14 regulates MMP-9 gene expression in OVCAR-4 ovarian cancer cells

PDF file - 418K, MMP-14 Ab inhibits invasion and metastasis in peritoneal ovarian cancer

PDF file - 62K

Wastewater treatment is the process of converting wastewater into water that is suitable to be discharged back into the environment. Proper wastewater treatment is essential in order to ensure the health of the population and the environment. According to a June 2012 USEPA case study in Fairfax County, Virginia, poorly designed/managed wastewater treatment systems lead to the pollution of nearly 900 miles of streams, lakes, and ponds. Due to environmental consequences resulting from poor management, two distinct treatment systems will be explored to observe their efficacy. They are aerobic digestion at Bland Correctional Center and oxidation ditch at Appalachian Detention Center. Both systems are common as they are simple and cost effective.My hypothesis is that aerobic digestion will be the more effective over oxidation ditch, and objective is to determine which is the more effective system. The tests to be performed include carbonaceous biochemical oxygen demand, total suspended solids, and E. coli. Once all data from both institutions are collected, it will all be analyzed and compared against current USEPA standards.It is anticipated that the Bland Correctional Center’s aerobic digestion system will have much lower E. coli, TSS, and CBOD readings in their samples because the institution sends samples to Blue Ridge Analytical Environmental Laboratory thrice weekly to ensure compliance to EPA standards, while Appalachian Detention Center only sends samples once monthly. Given the data observed so far, all tests conducted at Bland Correctional Center have proved that the facility is well within compliance of USPEA standards.

Graphical abstract, Highlights • Using surgical glue and a glove, we decompress and remove a large ovarian tumor. • The seal created by the Dermabond AdvancedTM prevents spillage of tumor contents. • The technique uses low cost materials that are found in most operating rooms. • The method has been successfully used on masses as large as 29 cm. • This method can be used when a large cyst needs to be drained without spillage., Large cystic ovarian tumors usually require surgical removal because of symptoms and the possibility of malignancy. The ideal surgical approach would minimize the risk of spillage of tumor contents while minimizing surgical morbidity. The present study aims to demonstrate a novel technique to drain large cystic ovarian tumors without spillage. A mini-laparotomy is performed and the tumor surface is exposed. Dermabond Advanced™ (USA Medical and Surgical Supplies 2019a) is applied to the tumor and a surgical glove (USA Medical and Surgical Supplies 2019b) is applied to the glue area. A small incision is made in the center of the portion of the glove that is adherent to the tumor. The cyst fluid is allowed to drain into the glove where it is suctioned away, collapsing the tumor. Once the tumor is sufficiently decompressed, it is exteriorized and resected with the glove still attached. The technique was initially developed in a pig model and subsequently successfully performed by mini-laparotomy on two patients with >20 cm ovarian masses. This novel technique uses inexpensive and readily available materials for draining large cystic ovarian tumors without spillage so that they can be removed via mini-laparotomy.

ImportancePatients with recurrent ovarian carcinoma frequently develop resistance to platinum-based chemotherapy, at which time treatment options become limited. ObjectiveTo evaluate the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor niraparib combined with pembrolizumab in patients with recurrent ovarian carcinoma. Design, Setting, and ParticipantsThe TOPACIO/KEYNOTE-162 (Niraparib in Combination With Pembrolizumab in Patients With Triple-Negative Breast Cancer or Ovarian Cancer) trial, an open-label, single-arm phases 1 and 2 study enrolled women with advanced or metastatic triple-negative breast cancer (TNBC) or recurrent ovarian carcinoma, irrespective of BRCA mutation status. Median follow-up was 12.4 months (range, 1.2 to >= 23.0 months). Data were collected from April 15, 2016, through September 4, 2018, with September 4, 2018, as a data cutoff, and analyzed from September 4, 2018, through January 30, 2019. InterventionsThe recommended phase 2 dose (RP2D) was 200 mg of oral niraparib once daily and 200 mg of intravenous pembrolizumab on day 1 of each 21-day cycle. Main Outcomes and MeasuresThe primary objectives of phase 1 were to evaluate dose-limiting toxic effects and establish the RP2D and dosing schedule. The primary objective of phase 2 was to assess objective response rate (ORR; complete plus partial responses). Results from the phase 1 ovarian carcinoma and TNBC cohorts and phase 2 ovarian carcinoma cohort are reported. Because of the similarity in the phase 1 and 2 ovarian carcinoma populations, the data were pooled to perform an integrated efficacy analysis. ResultsFourteen patients (9 with ovarian carcinoma and 5 with TNBC) in phase 1 and 53 patients with ovarian carcinoma in phase 2 were enrolled, for a pooled ovarian carcinoma cohort of 62 patients (median age, 60 years [range, 46-83 years]). In the integrated efficacy phases 1 and 2 ovarian carcinoma population (60 of 62 evaluable patients), ORR was 18% (90% CI, 11%-29%), with a disease control rate of 65% (90% CI, 54%-75%), including 3 (5%) with confirmed complete responses, 8 (13%) with confirmed partial responses, 28 (47%) with stable disease, and 20 (33%) with progressive disease. The ORRs were consistent across subgroups based on platinum-based chemotherapy sensitivity, previous bevacizumab treatment, or tumor BRCA or homologous recombination deficiency (HRD) biomarker status. Median duration of response was not reached (range, 4.2 to >= 14.5 months). At data cutoff, 2 patients with a response and 1 patient with stable disease continued to receive treatment. Conclusions and RelevanceNiraparib in combination with pembrolizumab is tolerable, with promising antitumor activity for patients with ovarian carcinoma who have limited treatment options regardless of platinum status, biomarker status, or prior treatment with bevacizumab. Responses in patients without tumor BRCA mutations or non-HRD cancers were higher than expected with either agent as monotherapy. Trial RegistrationClinicalTrials.gov identifier: NCT02657889

OBJECTIVE To describe the change over time in place of death (hospital, home, hospice) among all women in the United States who died of gynecologic malignancies and compare them with other leading causes of female cancer deaths. METHODS This is a retrospective cross-sectional study using national death certificate data from the Mortality Multiple Cause-of-Death Public Use Record Data. All women who died from gynecologic, breast, lung, and colorectal cancers were identified according to International Classification of Diseases, 10 Revision, cause of death from 2003 to 2015. Regression analyses with ordinary least-squares linear probability modeling were used to test for differences in location of death over time, and differences in trends by cancer type, while controlling for age, race, ethnicity, marital status, and education status. RESULTS From 2003 to 2015, 2,133,056 women died from gynecologic, lung, breast, and colorectal malignancies in the United States. A total of 359,340 died from gynecologic malignancies, including ovarian cancer (n=188,366 [52.4%]), uterine cancer (n=106,454 [29.6%]), cervical cancer (n=52,320 [14.6%]), and vulvar cancer (n=12,200 [3.4%]). Overall, 49.2% (n=176,657) of gynecologic cancer deaths occurred at home or in hospice. The relative increase from 2003 to 2015 in the rate of deaths at home or in hospice was 47.2% for gynecologic cancer deaths (40.5% in 2003 to 59.5% in 2015). In adjusted analyses, the trend in the percentage of deaths at home or in hospice increased at a rate of 1.6 percentage points per year for gynecologic cancer deaths (95% CI 1.5-1.6) vs 1.5 (95% CI 1.4-1.5, P

We report the design, synthesis, and biological evaluation of imidazopyridine-based peptidomimetics based on the substrate consensus sequence of Akt, an AGC family serine/threonine kinase hyperactivated in over 50% of human tumors. Our ligand-based approach led to the identification of novel substrate mimetic inhibitors of Akt1 featuring an unnatural extended dipeptide surrogate. Compound 11 inhibits Akt isoforms in the sub-micromolar range and exhibits improved proteolytic stability relative to a parent pentapeptide.

Most patients with ovarian cancer are diagnosed late in progression and often experience tumor recurrence and relapses due to drug resistance. Surface expression of matrix metalloprotease (MMP)-14 on ovarian cancer cells stimulates a tumor–stromal signaling pathway that promotes angiogenesis and tumor growth. In a cohort of 92 patients, we found that MMP-14 was increased in the serum of women with malignant ovarian tumors. Therefore, we investigated the preclinical efficacy of a MMP-14 monoclonal antibody that could inhibit the migratory and invasive properties of aggressive ovarian cancer cells in vitro. MMP-14 antibody disrupted ovarian tumor–stromal communication and was equivalent to Avastin in suppressing blood vessel growth in mice harboring Matrigel plugs. These effects on angiogenesis correlated with downregulation of several important angiogenic factors. Furthermore, mice with ovarian cancer tumors treated with anti–MMP-14 monotherapy showed a marked and sustained regression in tumor growth with decreased angiogenesis compared with immunoglobulin G (IgG)-treated controls. In a model of advanced peritoneal ovarian cancer, MMP-14–dependent invasion and metastasis was effectively inhibited by intraperitoneal administration of monoclonal MMP-14 antibody. Together, these studies provide a preclinical proof-of-concept for MMP-14 targeting as an adjuvant treatment strategy for advanced ovarian cancer. Cancer Res; 73(8); 2457–67. ©2013 AACR.

We previously reported that caffeoyl-amino acidyl-hydroxamic acid (CA-Xaa-NHOH) acted as both a good antioxidant and tyrosinase inhibitor, in particular when caffeic acid was conjugated with proline or amino acids having aromatic ring like phenylalanine. Here, various hydroxycinnamic acid (HCA) derivatives were further conjugated with phenylalanyl hydroxamic acid and prolyl hydroxamic acid (HCA-Phe-NHOH and HCA-Pro-NHOH) to study the structure and activity relationship as both antioxidants and tyrosinase inhibitors. When their biological activities were evaluated, all HCA-Phe-NHOH and HCA-Pro-NHOH exhibited enhanced antioxidant activity compared to HCA alone. Moreover, derivatives of caffeic acid, ferulic acid, and sinapic acid inhibited lipid peroxidation more efficiently than vitamin E analogue (Trolox). In addition, derivatives of caffeic acid and sinapic acid efficiently inhibited tyrosinase activity and reduced melanin content in melanocytes Mel-Ab cell.

Cases of women with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome developing leiomyomata are rare. A case with mitotically active leiomyomata has not previously been described to our knowledge.A 43-year-old woman with MRKH syndrome found to have an incidental pelvic mass on imaging studies underwent a diagnostic laparoscopy, followed by resection of leiomyomata and uterine remnant via mini laparotomy. Histopathology revealed focal infarction associated with a mitotically active area in one of the leiomyomata but with no evidence of marked cytologic atypia or hypercellularity. Focal adenomyosis was also noted.Studies have shown that mitotically active smooth cell tumors of the uterus having 5-9 mitoses/10 hpf and no cellular atypia have a metastatic rate too low to be regarded as sarcomas. Although the pathology findings in this case are benign with no need for continued surveillance by gynecologic oncology, regular follow-up with a gynecologist annually may be indicated for early diagnosis of recurrence secondary to the uncommon characteristics of this benign tumor, especially in this rare category of patients with Müllerian agenesis. Mitotically active leiomyomata can occur in patients with Müllerian agenesis, but the likelihood that a pelvic mass in a patient with MRKH syndrome is a sarcoma is extremely low.

Cooperativity with glucose is a key feature of human glucokinase (GK), allowing its crucial role as a glucose sensor in hepatic and pancreatic cells. We studied the changes in enzyme intrinsic tryptophan fluorescence induced by binding of different ligands to this monomeric enzyme using stopped-flow and equilibrium binding methods. Glucose binding data under pre-steady state conditions suggest that the free enzyme in solution is in a preexisting equilibrium between at least two conformers (super-open and open) which differ in their affinity for glucose (Kd* = 0.17 +/- 0.02 mM and Kd = 73 +/- 18 mM). Increasing the glucose concentration changes the ratio of the two conformers, thus yielding an apparent Kd of 3 mM (different from a Km of 7-10 mM). The rates of conformational transitions of free and GK complexed with sugar are slow and during catalysis are most likely affected by ATP binding, phosphate transfer, and product release steps to allow the kcat to be 60 s-1. The ATP analogue PNP-AMP binds to free GK (super-open) and GK-glucose (open) complexes with comparable affinities (Kd = 0.23 +/- 0.02 and 0.19 +/- 0.08 mM, respectively). However, cooperativity with PNP-AMP observed under equilibrium binding conditions in the presence of glucose (Hill slope of 1.6) is indicative of further complex tightening to the closed conformation. Another physiological modulator (inhibitor), palmitoyl-CoA, binds to GK with similar characteristics, suggesting that conformational changes induced upon ligand binding are not restricted by an active site ligand. In conclusion, our data support control of GK activity and Km through the ratio of distinct conformers (super-open, open, and closed) through either substrate or other ligand binding and/or dissociation.

Dipeptidyl peptidase-IV (DPP-IV) is a serine protease with a signature Asp-His-Ser motif at the active site. Our pH data suggest that Gly-Pro-pNA cleavage catalyzed by DPP-IV is facilitated by an ionization of a residue with a p K of 7.2 ± 0.1. By analogy to other serine proteases this p K is suggestive of His-Asp assisted Ser addition to the P1 carbonyl carbon of the substrate to form a tetrahedral intermediate. Solvent kinetic isotope effect studies yielded a D 2 O k cat / K m = 2.9 ± 0.2 and a D 2 O k cat = 1.7 ± 0.2 suggesting that kinetically significant proton transfers contribute to rate limitation during acyl intermediate formation (leaving group release) and hydrolysis. A “burst” of product release during pre steady-state Gly-Pro- p NA cleavage indicated rate limitation in the deacylation half-reaction. Nevertheless, the amplitude of the burst exceeded the enzyme concentration significantly (∼15-fold), which is consistent with a branching deacylation step. All of these data allowed us to better understand DPP-IV inhibition by saxagliptin (BMS-477118). We propose a two-step inhibition mechanism wherein an initial encounter complex is followed by covalent intermediate formation. Final inhibitory complex assembly ( k on ) depends upon the ionization of an enzyme residue with a p K of 6.2 ± 0.1, and we assigned it to the catalytic His-Asp pair which enhances Ser nucleophilicity for covalent addition. An ionization with a p K of 7.9 ± 0.2 likely reflects the P2 terminal amine of the inhibitor hydrogen bonding to Glu205/Glu206 in the enzyme active site. The formation of the covalent enzyme–inhibitor complex was reversible and dissociated with a k off of (5.5 ± 0.4) × 10 −5 s −1 , thus yielding a K i ∗ (as k off / k on ) of 0.35 nM, which is in good agreement with the value of 0.6 nM obtained from steady-state inhibition studies. Proton NMR spectra of DPP-IV showed a downfield resonance at 16.1 ppm. Two additional peaks in the 1 H NMR spectra at 17.4 and 14.1 ppm were observed upon mixing the enzyme with saxagliptin. Fractionation factors ( ϕ ) of 0.6 and 0.5 for the 17.4 and 14.1 ppm peaks, respectively, are suggestive of short strong hydrogen bonds in the enzyme–inhibitor complex.

Dipeptidyl peptidase-IV is a cell surface protease which plays an important role in glucose homeostasis through proteolytic inactivation of incretin hormones, primarily glucagon like peptide-1 (GLP-1). Substrate N-terminal amino acid (S2-S1) specificity is rather clearly defined, while no substantial information is available on the significance of amino acid interactions towards the C-terminus after the scissile bond (so called prime S1'-S4' or distant S5'-S28' sites). In the present study the increasing length of the peptide towards prime sites (S1'-S4') resulted in approximately 7-fold decrease in Km. Moreover, the Km for GLP-1 cleavage was comparable to that of an S2-S4' peptide, suggesting that few, if any, important enzyme-substrate interactions occur beyond the active site. Effect of substrate length on kcat was less obvious, but kcat/Km showed an increasing trend when His-Ala-pNA (representing the natural two N-terminal residues) was compared to GLP-1. To probe the impact of increasing substrate length on the free energy of activation (as has been suggested for elastase and chymotrypsin) we performed temperature studies. To adequately interpret thermodynamic data we sought to understand what steps limit the kcat expression. Steady-state parameters of the reactions catalyzed by serine proteases are composed of microscopic constants describing binding, acylation, and deacylation steps. Viscosity and pre-steady-state studies suggested that His-Ala-pNA cleavage is limited in the deacylation half-reaction, most likely the product release step. Thus, the free energy of activation, as calculated from the Eyring equation, is underestimated (at least for His-Ala-pNA) and the effect of substrate length on the acylation step (and transition-state stabilization) could not be unambiguously assessed.

The etiological agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus SARS-CoV. Similar to other coronaviruses, spike (S)-glycoprotein of the virus interacts with a cellular receptor and mediates membrane fusion to allow viral entry into susceptible target cells. Accordingly, S-protein plays an important role in virus infection cycle and is the primary target of neutralizing antibodies. To begin to understand its biochemical and immunological properties, we expressed both full-length and ectodomain of the protein in various primate cells. Our results show that the protein has an electrophoretic mobility of about 160–170 kDa. The protein is glycosylated with high mannose and/or hybrid oligosaccharides, which account for approximately 30 kDa of the apparent protein mass. The detection of S-protein by immunoassays was difficult using human convalescent sera, suggesting that the protein may not elicit strong humoral immune response in virus-infected patients. We were able to pseudotype murine leukemia virus particles with S-protein and produce SARS pseudoviruses. Pseudoviruses infected Vero E6 cells in a pH-independent manner and the infection could be specifically inhibited by convalescent sera. Consistent with low levels of antibodies against S-protein, neutralizing activity was weak with 50% neutralization titers ranging between 1:15 to 1:25. To facilitate quantifying pseudovirus-infected cells, which are stained blue with X-Gal, we devised an automated procedure using an ELISPOT analyzer. The high-throughput capacity of this procedure and the safety of using SARS pseudoviruses should make possible large-scale analyses of neutralizing antibody responses against SARS-CoV.

Objectives The aim of this study was to assess the feasibility of combining docetaxel, carboplatin, and gemcitabine (DoCaGem) as first-line treatment of ovarian and other mullerian origin tumors. Methods Four dose levels were planned for this phase I trial. Treatment consisted of carboplatin on day 1 only, docetaxel on days 1 and 8, and gemcitabine on days 1 and 8, every 21 days. Results Nineteen patients were enrolled, of whom 18 were evaluable for toxicity. The first 5 patients enrolled at dose level I (carboplatin AUC 5, 30 mg/m 2 docetaxel, 800 mg/m 2 gemcitabine) experienced no dose-limiting toxicity. At dose level II, 4/4 evaluable patients experienced significant bone marrow suppression that required dose reduction and/or dose delay. Further dose escalation was not attempted, and 9 additional patients were enrolled in dose level I. Of the 67 cycles administered on dose level I, day 8 treatment could not be administered due to bone marrow suppression in 16 cycles (24%), and prolongation of the cycle length from day 22 to day 29 was required in 13 cycles (19%). There were no episodes of febrile neutropenia in evaluable patients and no treatment-related deaths. Grade 3 nonhematologic toxicity (fatigue) occurred in 1 cycle. Response was determined in the 14 evaluable patients who tolerated 4 or more cycles of therapy, with 11 obtaining a complete clinical response and 3 obtaining a partial clinical response. Conclusions The DoCaGem regimen is highly active, but myelosuppression at dose level I prevents full dose delivery. Other strategies to combine these three active agents are reasonable to explore.

Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for β-site cleavage of APP, leading to the formation of the amyloid-β peptide that is thought to be pathogenic in Alzheimer’s disease (AD). Hence, BACE is an attractive pharmacological target, and numerous research groups have begun searching for potent and selective inhibitors of this enzyme as a potential mechanism for therapeutic intervention in AD. The mature enzyme is composed of a globular catalytic domain that is N-linked glycosylated in mammalian cells, a single transmembrane helix that anchors the enzyme to an intracellular membrane, and a short C-terminal domain that extends outside the phospholipid bilayer of the membrane. Here we have compared the substrate and active site-directed inhibitor binding properties of several recombinant constructs of human BACE. The constructs studied here address the importance of catalytic domain glycosylation state, inclusion of domains other than the catalytic domain, and incorporation into a membrane bilayer on the interactions of the enzyme active site with peptidic ligands. We find no significant differences in ligand binding properties among these various constructs. These data demonstrate that the nonglycosylated, soluble catalytic domain of BACE faithfully reflects the ligand binding properties of the full-length mature enzyme in its natural membrane environment. Thus, the use of the nonglycosylated, soluble catalytic domain of BACE is appropriate for studies aimed at understanding the determinants of ligand recognition by the enzyme active site.

It has been extremely difficult to elicit broadly cross-reactive neutralizing antibodies (Nabs) against human immunodeficiency virus type 1 (HIV-1). In this study, we compared the immunogenic properties of the wild-type and variable loop-deleted HIV-1 envelope glycoproteins. Mice were immunized with recombinant vaccinia viruses expressing either the wild-type or the variable loop-deleted (V1-2, V3, V4, and V1-3) HIV-1(DH12) gp160s. The animals were subsequently boosted with respective recombinant gp120s. All envelope constructs elicited similar levels of gp120-binding antibodies when analyzed by enzyme-linked immunosorbent assay (ELISA). However, the highest neutralizing activity was observed in sera from animals immunized with the wild-type envelope protein, followed by those immunized with DeltaV4 and DeltaV1-2. No neutralizing activity was detected in sera from animals immunized with DeltaV3 or DeltaV1-3. To identify immunogenic epitopes, ELISA was performed with overlapping 15-mer peptides that cover the entire length of gp120. For the wild-type gp120, the immunogenic epitopes mapped primarily to the variable loops V1-2 and to the conserved regions C1 and C5. When they were plotted onto known coordinates of gp120 core crystal structure, the epitopes in the conserved regions mapped predominantly to the inner domain of the protein. By immunizing with variable loop-deleted envelopes, the immune responses could be redirected to other regions of the protein. However, the newly targeted epitopes were neither on the exposed surface of the protein nor on the receptor binding regions. Interestingly, the removal of the V3 loop resulted in loss of immunoreactivity for both V3 and V1/V2 loops, suggesting structural interaction between the two regions. Our results suggest that obtaining broadly reactive Nabs may not be achieved simply by deleting the variable loops of gp120. However, the observation that the immune responses could be redirected by altering the protein composition might allow us to explore alternative strategies for modifying the antigenic properties of HIV-1 envelope glycoprotein.

A highly endo selective Diels–Alder reaction provides efficient access to the substituted cis -dehydrodecalin core structure of meiogynin A. Elaboration into the fully functionalized scaffold was achieved in three steps from cycloadduct 20a . This strategy is amenable to the synthesis of diversely substituted meiogynin analogs for the discovery of novel inhibitors of Bcl-xL and related anti-apoptotic proteins.

The great difficulty in eliciting broadly cross-reactive neutralizing antibodies (NAbs) against human immunodeficiency virus type 1 (HIV-1) isolates has been attributed to several intrinsic properties of their viral envelope glycoprotein, including its complex quaternary structure, extensive glycosylation, and marked genetic variability. Most previously evaluated vaccine candidates have utilized envelope glycoprotein from a single virus isolate. Here we compare the breadth of NAb and protective immune response following vaccination of pigtailed macaques with envelope protein(s) derived from either single or multiple viral isolates. Animals were challenged with Simian/human immunodeficiency virus strain DH12 (SHIV DH12 ) following priming with recombinant vaccinia virus(es) expressing gp160(s) and boosting with gp120 protein(s) from (i) LAI, RF, 89.6, AD8, and Bal (Polyvalent); (ii) LAI, RF, 89.6, AD8, Bal, and DH12 (Polyvalent-DH12); (iii) 89.6 (Monovalent-89.6); and (iv) DH12 (Monovalent-DH12). Animals in the two polyvalent vaccine groups developed NAbs against more HIV-1 isolates than those in the two monovalent vaccine groups ( P = 0.0054). However, the increased breadth of response was directed almost entirely against the vaccine strains. Resistance to SHIV DH12 strongly correlated with the level of NAbs directed against the virus on the day of challenge ( P = 0.0008). Accordingly, the animals in the Monovalent-DH12 and Polyvalent-DH12 vaccine groups were more resistant to the SHIV DH12 challenge than the macaques immunized with preparations lacking a DH12 component (viz. Polyvalent and Monovalent-89.6) ( P = 0.039). Despite the absence of any detectable NAb, animals in the Polyvalent vaccine group, but not those immunized with Monovalent-89.6, exhibited markedly lower levels of plasma virus than those in the control group, suggesting a superior cell-mediated immune response induced by the polyvalent vaccine.

Objectives. The goals of this study were to determine the length of stay (LOS) after abdominal surgery following implementation of practice guidelines on a gynecologic oncology service, to identify adverse outcomes of early discharge, and to identify clinical predictors of longer LOS. Methods. A retrospective chart review of 266 consecutive patients who had elective abdominal surgery was performed. Clinical data, LOS, and follow-up data were abstracted. Univariate and multivariate analyses were performed to identify clinical variables predictive of LOS. Results. Mean LOS was 2.94 days. Seven (2.6%) patients were readmitted after discharge. With multivariate analysis, extensive surgical procedures, coronary artery disease, and bowel surgery were predictive of longer LOS ( P Conclusions. Early discharge following abdominal surgery was possible for most patients and was associated with a low rate of readmission. Extensive surgical procedures, coronary artery disease, and bowel surgery were predictive of longer LOS.

SummaryHuman platelets express two distinct G protein-coupled ADP receptors, one coupled to phospholipase C through Gq, P2Y1, and the other to inhibition of adenylyl cyclase through Gi, P2TAC. We have recently shown that concomitant intracellular signaling from both the P2TAC and P2Y1 receptors is essential for ADP-induced platelet aggregation. Previous studies have tested whether ADP causes a decrease in the basal cAMP level and this reduction promotes platelet aggregation, but did not study the effect of decreased cAMP levels when the Gq pathway is selectively activated. Since we are now aware that platelet aggregation requires activation of two receptors, we investigated whether the function of P2TAC receptor activation, leading to inhibition of platelet adenylyl cyclase, could be replaced by direct inhibition of adenylyl cyclase, when Gq pathway is also activated, a possibility that has not been addressed to date. In the present study, we supplemented the P2Y1 mediated Gq signaling pathway with inhibition of the platelet adenylyl cyclase by using SQ22536 or dideoxyadenosine, or by selective activation of the α2A adrenoceptors with epinephrine. Although SQ22536, dideoxyadenosine, and epinephrine reduced the cAMP levels, only epinephrine could mimic the P2TAC receptor mediated signaling events, suggesting that reduction in basal cAMP levels does not directly contribute to ADP-induced platelet activation. Adenosine-5’-phosphate-3’-phosphosulfate, a P2Y1 receptor antagonist, completely blocked ADP-induced inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate formation suggesting that P2TAC-mediated activation of Gi (or other G proteins) does not activate phospholipase C. These results suggest that a signaling event downstream from Gi, independent of the inhibition of platelet adenylyl cyclase, contributes to αIIbβ3 activation.

We propose a systematic approach to quantify the impact of nonuniform traffic on the performance of non-blocking switches with output queueing. We do so in the context of a simple queueing model where cells arrive to input ports according to independent Bernoulli processes, and are switched to an output port under a random routing mechanism. We give conditions on pairs of input rate vectors and switching matrices which ensure various stochastic comparisons for performance measures of interest. These conditions are formulated in terms of the majorization ordering while the comparison results are expressed in the strong and convex increasing orderings

Objective: To estimate the incidence of dysplasia in patients with Papanicolaou smears showing atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (SIL) and to identify clinical predictors of dysplasia in these patients. Methods: Patients referred for ASCUS and low-grade SIL were reviewed retrospectively. All patients were evaluated with immediate colposcopy. A multivariate logistic regression analysis was performed to identify clinical predictors of histologic SIL and histologic high-grade SIL. Results: One hundred thirty-seven (34%) of 406 consecutive patients had histologic SIL. Regression analysis identified age (under 35 versus 35 years or above) and initial smear (low-grade SIL versus ASCUS) as statistically significant predictors of histologic SIL and high-grade SIL (P < .001). When patient outcomes were analyzed by age and initial Papanicolaou smear results, the subgroup of patients 35 years or older with ASCUS had low incidences of histologic SIL (14%) and high-grade SIL (1%). The other subgroups (under 35 years with ASCUS, under 35 years with low-grade SIL, and 35 years or older with low-grade SIL) had incidences of histologic SIL and histologic high-grade SIL of at least 28% and 14%, respectively. Conclusion: The high incidence of dysplasia in patients with minimally abnormal Papanicolaou smears suggests that immediate colposcopy might be appropriate for many of these patients. Age and initial Papanicolaou smear are predictive of dysplasia and might be used to select patients who have low incidence of dysplasia and might not require immediate colposcopy.

Context.—Adequacy criteria for endometrial biopsy samples do not exist. Objective.—To assess the sensitivity of endometrial sampling for detecting neoplasia in the setting of extensive glandular and stromal breakdown. Design.—Retrospective case-control study. Surgical pathology records between 1996 and 2005 at Beth Israel Deaconess Medical Center (Boston, Mass) were searched for endometrial samples with diagnoses containing the key words “menstrual” or “extensive breakdown.” Hospital records for these women were parsed for demographics, clinical indications, and follow-up with rebiopsy within 6 months. Age cutoffs enriched the population for women at higher risk for carcinoma. A control group, consisting of 2 age-matched control patients for each test patient, was also studied; each control patient had an endometrial sample taken within a 6-month period and was not diagnosed with extensive breakdown, menstrual endometrium, or neoplasia on initial sampling. Results.—Fifty-four cases were identified. The primary biopsy reports had benign descriptive diagnoses (ie, proliferative, secretory, polyp). Follow-up biopsies showed benign pathology in all cases and specific causes of bleeding—including polyp, leiomyoma, or endometritis—in 28 (52%) of 54. In the control group, neoplasia was found in 2 of the 108 follow-up biopsies. Only 5 other controls had specific diagnoses; all were polyps. Conclusions.—Extensive breakdown or menstrual-pattern endometrium may mask other specific benign pathologies but does not commonly mask cancer.

BACKGROUND The standard treatment for endometrial carcinoma is staging laparotomy with total abdominal hysterectomy and bilateral salpingo-oophorectomy. In an attempt to preserve childbearing potential, selected patients with endometrial carcinoma were treated with progestin alone as primary therapy. METHODS Patients were identified through searches of tumor registries and solicitation of consulting gynecologic oncologists at the affiliated institutions of the University of California-Los Angeles Center for the Health Sciences. Only those patients with a diagnosis of endometrial carcinoma treated with progestin alone as primary therapy were included in the study. Independent pathologic review was performed by a recognized expert gynecologic pathologist to exclude cases of endometrial hyperplasia. A MEDLINE search was conducted to identify reports of similarly treated patients. RESULTS Seven patients were treated with progestin alone for endometrial carcinoma at the study institution. Fourteen additional patients were identified through the literature search. Combining the data for all patients, 13 of 21 patients (62%) had an initial response to progestins. Three initial responders later developed recurrent disease, one of whom was found to have extrauterine disease at laparotomy. Eight of 21 patients (38%) did not respond to progestins and underwent more definitive treatment. None of these patients later developed recurrent disease. Six viable infants were delivered of three patients after therapy. Nineteen of 21 patients were alive without evidence of disease at last follow-up. CONCLUSIONS The results of this study show that premenopausal women with endometrial carcinoma may be treated successfully with progestin therapy alone as primary therapy to preserve childbearing potential. Cancer 1997; 79:320-7. © 1997 American Cancer Society.

Objective: To determine whether the expression of vascular endothelial growth factor (VEGF) is altered by treatment of an in vivo tumor with 17β-estradiol (E2) or medroxyprogesterone acetate (MPA). Methods: A well-differentiated endometrial carcinoma tumor was isolated from a patient and explanted into the dorsal skin of ovariectomized nude mice, from which it was serially passaged in vivo. The explanted tumor retained all the properties of the original tumor, including estrogen and progesterone receptor expression and growth promotion and inhibition by E2 and MPA, respectively. The mice were treated with continuous E2 administration followed by treatment with either a single intramuscular administration of 2 mg MPA or weekly administrations of 2 mg MPA. Untreated tumor-bearing mice served as controls. The tumors were harvested at 0 to 21 days from first MPA administration. RNA from the tumors was isolated and VEGF expression was determined by Northern analysis. Results: VEGF was expressed in the absence of treatment with E2 or MPA, and expression was unaltered by continuous treatment with E2. Additional treatment with a single dose of MPA did not alter expression at Days 1, 2, 3, 7, 14, and 21, and additional treatment with weekly doses of MPA did not alter expression at Weeks 1, 2, and 3. Conclusions: VEGF is constitutively expressed in this in vivo model of endometrial carcinoma, and its expression is unaltered by treatment with E2 or E2 + MPA. Regulation of VEGF expression is not a mechanism by which these hormones exert their growth effects on endometrial tumors.

Mcl-1, an anti-apoptotic member of the Bcl-2 protein family, is overexpressed in a broad range of human cancers and plays a critical role in conferring resistance to chemotherapy. In the course of screening a natural product-like library of sesquiterpenoid analogs, we identified substituted hexahydronaphthalenes that showed activity against the Mcl-1/BimBH3 interaction in vitro. Here, we describe the synthesis of a small library of analogs and their biological evaluation. The most potent inhibitor in the series (19) exhibits an IC(50) of 8.3 μM by ELISA and disrupts the interaction between endogenously expressed Mcl-1 and Bim in cultured MDA-MB-468 breast cancer cells.

Reports on the usefulness of serum markers for predicting liver necroinflammation are limited. The aim of this study was to determine the serum markers that predict significant inflammation in patients with chronic hepatitis B (CHB) and C (CHC) and normal or mildly elevated serum aminotransferase levels.Two hundred twenty-seven patients with CHB or CHC with normal or mildly elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (≤60 IU/L) were enrolled in this study. Significant inflammation was defined as inflammatory grade ≥3 activities using the Batt-Ludwig scoring system. The correlation between liver histology and serum markers of liver inflammation was analysed.Forty-eight (21.1%) and eight patients (3.5%) had grade 3 and 4 inflammation respectively. Univariate analysis revealed that age, platelet coun, and AST, ALT, γ-glutamyl transpeptidase, alkaline phosphatase, hyaluronic acid, haptoglobin, apolipoprotein A1 and procollagen III N-terminal peptide levels were significantly different between the patients with and without significant inflammation. There were no significant differences in the cytokeratin-18 fragment levels between the two groups. On the basis of multivariate analysis, the AST and apolipoprotein A1 levels and stage of fibrosis were highly predictive of significant inflammation. Using AST and apolipoprotein cut-off values ≥44 IU/L and ≤100 ng/ml, respectively, the presence of significant inflammation was predicted with high specificity (96.5%) and with a negative predictive value of 76.3%.The AST and apolipoprotein A1 levels were shown to be independent predictors of significant inflammatory activities in patients with CHB and CHC and normal or mildly elevated aminotransferase levels.

The harmonic balance (HB) with alternating frequency time (AFT) method is applied to a single-degree-of-freedom (SDOF) system with piecewise-smooth non-linearity. The periodic solution of the system with the discontinuous type non-linearity is obtained by discretizing the non-linear function in the time domain. The explicit form of the Jacobian matrix is derived using a Galerkin technique to ensure the robustness, convergence and computational efficiency of the algorithm during numerical iteration. An approximate, but accurate stability analysis for the determined periodic solution is achieved through a Floquet analysis based on numerical integration for one period. The concept of the hyperbolicity in a Poincare map is used to determine the stability as well as the type of bifurcation associated with a determined periodic solution. The numerical simulation shows that two types of chaotic motion can exist: one through period doubling and the other due to boundary crisis.

Four hundred patients admitted to a gynecologic oncology service for laparotomy through a midline abdominal incision underwent wound closure using a continuous single-layer technique. Most...

A new HB (Harmonic Balance)/AFT (Alternating Frequency Time) method is further developed to obtain synchronous and subsynchronous whirling response of nonlinear MDOF rotor systems. Using the HBM, the nonlinear differential equations of a rotor system can be transformed to algebraic equations with unknown harmonic coefficients. A technique is applied to reduce the algebraic equations to only those of the nonlinear coordinates. Stability analysis of the periodic solutions is performed via perturbation of the solutions. To further reduce the computational time for the stability analysis, the reduced system parameters (mass, damping, and stiffness) are calculated in terms of the already known harmonic coefficients. For illustration, a simple MDOF rotor system with a piecewise-linear bearing clearance is used to demonstrate the accuracy of the calculated steady-state solutions and their bifurcation boundaries. Employing ideas from modern dynamics theory, the example MDOF nonlinear rotor system is shown to exhibit subsynchronous, quasi-periodic and chaotic whirling motions.

Alzheimer's disease (AD) is a progressive neurodegenerative disease. Amyloid beta (Abeta) peptides are hypothesized to cause the initiation and progression of AD based on pathologic data from AD patients, genetic analysis of mutations that cause early onset forms of AD, and preclinical studies. Based on this hypothesis, beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) inhibitors are an attractive therapeutic approach for AD because cleavage of the APP by BACE1 is required to form Abeta. In this study, three potent BACE1 inhibitors are characterized. All three inhibitors decrease Abeta formation in cultured cells with IC(50) values less than 10 nM. Analysis of APP C-terminal fragments by immunoblotting and Abeta peptides by mass spectrometry showed that these inhibitors decreased Abeta by inhibiting BACE1. An assay for Abeta1-40 in mice was developed and used to show that these BACE1 inhibitors decreased plasma Abeta1-40, but not brain Abeta1-40, in wild-type mice. Because these BACE1 inhibitors were substrates for P-glycoprotein (P-gp), a member of the ATP-binding cassette superfamily of efflux transporters, these inhibitors were administered to P-gp knockout (KO) mice. These studies showed that all three BACE1 inhibitors decreased brain Abeta1-40 in P-gp KO mice, demonstrating that P-gp is a major limitation for development of BACE1 inhibitors to test the amyloid hypothesis. A comparison of plasma Abeta1-40 and brain Abeta1-40 dose responses for these three compounds revealed differences in relative ED(50) values, indicating that factors other than P-gp can also contribute to poor brain activity by BACE1 inhibitors.

Objective The purpose of this study was to quantify, categorize, and illustrate discrepancies between preoperative radiologic, surgical, and pathologic diagnoses and to assess the potential impact of discrepancies on clinical care. Methods Adnexal masses reported by pathology during a 16-month period were included if prior imaging at our institution had been performed. Up to 3 sonographic, computed tomographic, and magnetic resonance imaging examinations were reviewed by a gynecologic sonographer and compared with the reported pathologic findings. Cases in which ambiguities were not resolved by consulting the surgical notes were reviewed by a gynecologic pathologist, who confirmed or modified the diagnosis and assigned a score to the pathology quality assurance issue: 0, no pathology quality assurance error; 1, differences in terminology; 2, discrepancy of diagnostic interpretation, benign findings; or 3, discrepancy of diagnostic interpretation, malignant findings. Results Of 418 total masses, there was a discrepancy between imaging and pathology in 73 (17%) masses and 68 (21%) pathology reports. Twenty-five (6%) had pathology discrepancies resolved by correlation with the surgical notes alone (eg, torsion seen during surgery but not evident on pathologic examination). Histologic review was performed for 48 (11%) of 418 masses, with pathology errors identified in 34 (71%) of 48. Quality assurance scores were 0 (n = 14), 1 (n = 14), and 2 (n = 20), with no cases receiving a score of 3. Examples of pathology errors included gross (complex versus simple) and microscopic (neoplastic versus functional versus mesothelial) characterization of cysts, sizes of lesions not being described, characterization of fibrous lesions (cystadenofibroma versus cystadenoma), and lack of correlation with imaging (lesions not described). Conclusions This study illustrates the importance of imaging, surgical, and histologic correlation in assessing the diagnostic accuracy of sonography of adnexal masses.

A HB (Harmonic Balance)/AFT (Alternating Frequency/Time) technique is developed to obtain synchronous and subsynchronous whirling motions of a horizontal Jeffcott rotor with bearing clearances. The method utilizes an explicit Jacobian form for the iterative process which guarantees convergence at all parameter values. The method is shown to constitute a robust and accurate numerical scheme for the analysis of two dimensional nonlinear rotor problems. The stability analysis of the steady-state motions is obtained using perturbed equations about the periodic motions. The Floquet multipliers of the associated Monodromy matrix are determined using a new discrete HB/AFT method. Flip bifurcation boundaries were obtained which facilitated detection of possible rotor chaotic (irregular) motion as parameters of the system are changed. Quasi-periodic motion is also shown to occur as a result of a secondary Hopf bifurcation due to increase of the destabilizing cross-coupling stiffness coefficients in the rotor model.

Adenosine diphosphate (ADP) is an important platelet agonist, causing the shape change and aggregation required for physiological hemostasis. We have recently demonstrated that the P2Y1 receptor plays an important role in ADP-induced shape change and aggregation in human platelets. The role of the P2Y1 receptor in these physiological responses can be conclusively delineated with gene-knockout approaches in transgenic mice. However, before proceeding to the P2Y1 gene-knockout mice generation, it is important to demonstrate that the P2Y1 receptor plays an essential role in ADP-induced shape change and aggregation in mouse platelets. We examined platelets pooled from twenty 129J mice, a strain used in the generation of knockout mice. Immunofluorescence experiments using P2Y1 specific antiserum detected the presence of the P2Y1 receptor on mouse platelets. ARL 66096, a potent P2T(AC) receptor antagonist, caused a dose-dependent inhibition of both ADP-induced aggregation and ADP-induced inhibition of adenylyl cyclase, without affecting shape change or calcium mobilization. On the other hand, adenosine-2'-phosphate-5'-phosphate (A2P5P), a P2Y1 receptor-selective antagonist, caused a dose-dependent inhibition of ADP-induced aggregation and shape change, as well as inhibiting the mobilization of calcium from intracellular stores. A2P5P had no effect on the inhibition of adenylyl cyclase by ADP. These findings clearly demonstrate the existence of two distinct ADP receptors, the P2Y1 and P2T(AC), in mouse platelets with similar function as in human platelets.

NTRAVENOUS LEIOMYOMA is a rare benign tumor of the uterus extending into the right side of the heart through the inferior vena cava (IVC) in 10% of patients. It commonly presents in postmenopausal parous women. 1 This tumor manifests itself clinically with menstrual alteration, dysmenorrhea, ascites, Budd Chiari syndrome, pelvic masses, right heart failure, syncope, and sometimes sudden death, caused by obstruction of the IVC or of the tricuspid valve. The common differential diagnoses for this type of tumor include cardiac tumor or thrombus in transit. 2 In this case, the authors believe that the transesophageal echocardiogram (TEE) examination and the finding of no intracardiac tethering of the tumor allowed the surgery to be performed as a 1-stage procedure and permitted the surgeons to use only a laparotomy incision. Intraoperative TEE is a powerful tool to help plan the surgical approach and evaluate the completeness of surgical excision in patients with intracavocardiac leiomyomatosis. CASE REPORT A 56-year-old woman presented with progressive fatigue for several weeks and difficulty in breathing for 2 weeks. Her past medical history was significant for postmenopausal uterine bleeding 1 year before with negative ultrasound imaging and without a palpable mass on physical examination. She had a history of hypertension, asthma, and nasal polyps. She had a 40 pack per year history of smoking and no history of alcohol use. An ultrasound of the gallbladder suggested a clot in the IVC. A subsequent computed tomography scan of the abdomen and chest revealed a pelvic mass and possible clot extension into the IVC. She was referred from the outside hospital for further care and management. Her initial examination revealed no acute distress. She was 165 cm tall and weighed 125 kg. Her vital signs were a heart rate of 82 beat/min, blood pressure of 150/70 mmHg, a respiratory rate of 14 breaths/min, and 95% oxygen saturation while on oxygen by nasal cannula at 2 L/min, and a room air saturation of 92%. Her laboratory results revealed elevated liver enzymes (alanine aminotransferase 165 U, alanine aminotransferase 149 U), a normal bilirubin level, electrolytes, and hematocrit. She was admitted to the intensive care unit (ICU) and placed on continuous hemodynamic monitoring, oxygen therapy and continuous intravenous heparin. A chest x-ray and electrocardiogram were normal. The abdominal computed tomography scan, chest x-ray, and magnetic resonance imaging showed a uterine mass extending through the right gonadal vein into the IVC to the level of the IVC/right atrium (RA) junction as well as extension into the proximal left renal vein. The initial TEE performed in the ICU showed a polycystic mass measuring 1.8 cm at its greatest visible diameter at the junction of the IVC and RA. The mass protruded slightly into the RA from the IVC with turbulent flow seen around the mass. All the chambers were of normal size with no interseptal defects and normal-appearing valves with mild mitral regurgitation. Biventricular systolic function was normal. There was no pericardial effusion. She was scheduled for exploratory laparotomy, with preparation for median sternotomy, cardiopulmonary bypass, and total circulatory arrest, if necessary. In the holding area, a large-bore peripheral intravenous catheter and a radial arterial catheter were started. After instituting the standard American Society of Anesthesiology monitors and arterial pressure monitoring, she was anesthetized with propofol and endotracheal intubation was facilitated with pancuronium. Anesthesia was maintained with isoflurane in 100% oxygen and intermittent fentanyl for analgesia. After suctioning her stomach with an orogastric tube, the TEE probe was introduced and used to visualize the wire for left internal jugular cannulation, taking care not to dislodge the tumor in the RA. An 8F introducer was placed through her left internal jugular. TEE confirmed the presence of a polycystic, serpentine elongated, nonhomogenous echogenic tumor extending from the hepatic and suprahepatic IVC into the RA with no apparent atrial or IVC attachments (Fig 1). Her hepatic veins, right ventricle, and pulmonary arteries did not reveal any tumor. Her chest, abdomen, and groin were sterilely prepared for the surgery. The cardiac surgical team, along with a cardiopulmonary bypass machine, were available on standby. The initial portion of the procedure involved a lower midline laparotomy and dissection of the tumor by the gynecologic oncologists and vascular surgeons. The vena cava was mobilized so that a proximal clamp could be placed. During this mobilization, the intracardiac portion of the tumor was continuously monitored using TEE, and the authors noted that the tumor moved back into the hepatic IVC by 5 cm. This movement suggested that the tumor could be removed through the IVC (Fig 2). The dissection of the uterus and gonadal vein was completed. The cava was then further mobilized with a clamp on the IVC and with hand control of the vena cava above the gonadal vein. A venotomy was made on the anterior wall of the gonadal vein, to which the tumor was attached, extending to the vena cava, and the tumor was resected free circumferentially at the entrance to the vena cava. Beyond that, the tumor was not attached to the wall of the vena cava, and, with TEE guidance, it was possible to remove the entire tumor through the venotomy in the abdomen. The patient was volume resuscitated during this period. The IVC was noted to be completely clear of tumor, and there was no residual turbulent flow in the IVC. Radical abdominal hysterectomy with bilateral salpingo-oophorectomy was completed. The frozen section on the tumor indicated that it was benign. Further pathology revealed a small endometrial cancer in addition to the intravascular tumor. The patient was left intubated overnight and monitored in the ICU. She had an uneventful recovery and was discharged home on the fourth postoperative day.

To establish a baseline for monitoring resistance mutation to protease inhibitors (PI), we determined protease(PR) sequences in peripheral blood mononuclear cells obtained from 43 PI-naive Korean HIV-1 infected patients. Interestingly, phylogenetic analysis revealed that 41 of the sequences belonged to subtype B, one belonged to subtype A, and one was unique, not clustering with any subtype. Thirty-one (76%) of the 41 sub-type B sequences formed a subclade within subtype B, a so-called "Korean B cluster." Polymorphisms were observed at 34 (34.3%) of the PR codons. One patient (2.3%) harbored a primary resistance-conferring mutation, L90M along with L63P and A71V, and all 43 strains showed some secondary associated with drug resistance. The percentage of patients with 7, 5, 4, 3, 2, and 1, resistance mutations were 2%, 2%, 14%, 23%,37%, and 9%, respectively. A novel polymorphism in subtype B, L63M was detected in two patients. Another patient showed a gross deletion (257 bp) after codon 91. The average genetic distance of the 41 subtype B sequences to the HXB2 sequence was 3.0% (range, 1.0-5.1%). Six hemophiliacs were infected with a domestic strain of HIV-1 subtype B, while the other two hemophiliacs were infected with nondomestic subtype B and had lived outside Korea. Although this is the first report on the molecular nature of PR in Korea, there is also a need to establish baselines for nonsubtype B HIV-1.

An effective vaccine against the human immunodeficiency virus type 1 (HIV-1) will very likely have to elicit both cellular and humoral immune responses to control HIV-1 strains of diverse geographic and genetic origins. We have utilized a pathogenic chimeric simian-human immunodeficiency virus (SHIV) rhesus macaque animal model system to evaluate the protective efficacy of a vaccine regimen that uses recombinant vaccinia viruses expressing simian immunodeficiency virus (SIV) and HIV-1 structural proteins in combination with intact inactivated SIV and HIV-1 particles. Following virus challenge, control animals experienced a rapid and complete loss of CD4 + T cells, sustained high viral loads, and developed clinical disease by 17 to 21 weeks. Although all of the vaccinated monkeys became infected, they displayed reduced postpeak viremia, had no significant loss of CD4 + T cells, and have remained healthy for more than 15 months postinfection. CD8 + T-cell and neutralizing antibody responses in vaccinated animals following challenge were demonstrable. Despite the control of disease, virus was readily isolated from the circulating peripheral blood mononuclear cells of all vaccinees at 22 weeks postchallenge, indicating that immunologic control was incomplete. Virus recovered from the animal with the lowest postchallenge viremia generated high virus loads and an irreversible loss of CD4 + T-cell loss following its inoculation into a naïve animal. These results indicate that despite the protection from SHIV-induced disease, the vaccinated animals still harbored replication-competent and pathogenic virus.

The oncoprotein hdm2 ubiquitinates p53, resulting in the rapid degradation of p53 through the ubiquitin (Ub)–proteasome pathway. Hdm2-mediated destabilization and inactivation of p53 are thought to play a critical role in a number of human cancers. We have used an in vitro enzyme assay, monitoring hdm2-catalyzed Ub transfer from preconjugated Ub-Ubc4 to p53, to identify small molecule inhibitors of this enzyme. Three chemically distinct types of inhibitors were identified this way, each with potency in the micromolar range. All three types of compounds display selective inhibition of hdm2 E3 ligase activity, with little or no effect on other Ub-using enzymes. Most strikingly, these compounds do not inhibit the autoubiquitination activity of hdm2. Steady-state analysis reveals that all three classes behave as simple reversible inhibitors of the enzyme and that they are noncompetitive with respect to both substrates, Ub-Ubc4 and p53. Studies of the effects of combinations of two inhibitory molecules on hdm2 activity indicate that the three types of compounds bind in a mutually exclusive fashion, suggesting a common binding site on hdm2 for all of these inhibitors. These compounds establish the feasibility of selectively blocking hdm2-mediated ubiquitination of p53 by small molecule inhibitors. Selective inhibitors of hdm2 E3 ligase activity could provide a novel mechanism for the development of new chemotherapeutics for the treatment of human cancers.

Neutralizing antibody (NAb) is a critical component of an immune system that can potentially provide sterilizing protection against human immunodeficiency virus type 1 (HIV-1). Therefore, an in vitro assay that can rapidly, safely, and accurately evaluate the NAb response vaccine candidates elicit, especially against a large number of HIV-1 variants, would be highly valuable. It has been demonstrated that HIV-1 envelope glycoprotein lacking the cytoplasmic domain can pseudotype murine leukemia virus encoding the beta-galactosidase gene and that this pseudovirus can specifically infect CD4(+) cells (Schnierle BS, Stitz J, Bosch V, et al.: Proc Natl Acad Sci USA 1997;94:8640-8645). Because the pseudovirus is not biohazardous and because the infection can be quantitatively determined within 2 days, we examined the feasibility of using the pseudovirus for high-throughput neutralization assays for HIV-1. We have generated viruses pseudotyped with gp140 of six different HIV-1 isolates (LAI, RF, Bal, AD8, 89.6, and DH12). All six pseudoviruses were infectious and exhibited expected coreceptor usage phenotype in HOS-CD4 cells expressing either CCR5 or CXCR4. More importantly, the neutralization sensitivity profile of these pseudoviruses was virtually identical to that observed from more conventional neutralization assays using either HIV-1 or SHIV. All pseudoviruses could be neutralized by broadly reactive human monoclonal antibody IgG1 b12. Our results indicate that the pseudoviruses are ideal for high-throughput evaluation of immune sera for their capacity to broadly neutralize a large number of HIV-1 isolates.

The mdm2 gene product is an important regulator of p53 function and stability. mdm2 is an E3 ubiquitin ligase for p53 and the RING finger domain of mdm2 is critical for ligase activity. Ubiquitin (Ub) conjugation is a general targeting modification and poly-ubiquitin chains specifically target proteins to the proteasome for degradation. In this report, we show that the multistep cascade of mdm2-mediated p53 ubiquitination can be reduced to three purified recombinant proteins: ubiquitin-conjugated E2, mdm2, and p53. This simplification allows enzymatic analysis of the isolated ligase reaction. The simplified reaction recapitulates the ubiquitination of p53 observed with individual components and the p53-Ub((n)) is qualitatively similar to p53-Ub((n)) detected in lactacystin-treated cells. Surprisingly, we find that p53 is modified with multiple mono-ubiquitin moieties as opposed to a poly-ubiquitin chain. Finally, kinetic analysis indicates the transfer reaction proceeds either through a modified Ping Pong mechanism involving requisite enzyme isomerization steps, or through a Rapid Equilibrium Random Bi Bi mechanism involving very large anti-cooperative interactions between the two substrate binding pockets on the enzyme, mediated through allosteric changes in enzyme structure.

Objectives. The goal of this study was to determine the maximally tolerated doses (MTDs) of carboplatin, paclitaxel (Taxol), etoposide, and cyclophosphamide (CTEC) with granulocyte-colony stimulating factor (G-CSF, Filgrastim) support as first-line chemotherapy in women with advanced epithelial ovarian cancer (EOC). Methods. Newly diagnosed patients with either stage IV EOC, or stage III EOC and any amount of gross residual tumor after surgical debulking were eligible to receive six cycles of CTEC over five different dose levels in this phase I trial (planned 21-day cycle length). Paclitaxel, carboplatin, and cyclophosphamide were administered intravenously on Day 1, and oral etoposide was administered on Days 1, 2, and 3. G-CSF was administered beginning Day 4. Results. Twenty patients received a total of 98 cycles of CTEC over the five dose levels evaluated. Bone marrow suppression was the major toxic effect, with grade 4 neutropenia and thrombocytopenia being observed in 25 and 23% of cycles, respectively. The overall incidence of febrile neutropenia was 10%, and no toxic deaths occurred. No grade IV thrombocytopenia or febrile neutropenia was observed once the carboplatin dose was reduced from AUC of 7 to 5. Nonhematologic toxicity was generally mild (grade 2 or less). Dose-limiting toxicity was not observed at the highest dose level evaluated in this study, preventing assignment of the MTD. The clinical complete response rate was 92%, although 15 of 16 evaluable patients have progressed with a median progression-free interval of 4 months (range, 2–11 months). One patient remains disease-free 9 months from the completion of CTEC. Conclusions. The CTEC regimen is well tolerated and highly active. Although the MTD was not reached in this study, the short median progression-free interval suggests that this regimen is unlikely to be superior to standard treatment with paclitaxel and carboplatin. Strategies to optimize the development of future combination chemotherapy regimens in the treatment of newly diagnosed ovarian cancer are discussed.

Objective. The objective of this study was to determine whether hysteroscopy improved upon the diagnostic sensitivity of dilatation and curettage (D+C) in the detection of endometrial hyperplasia and carcinoma. Methods. A retrospective chart review was conducted of all patients undergoing hysteroscopy/D+C for abnormal uterine bleeding between 1991 and 1995. Hysteroscopic impressions and D+C diagnoses were compared. Results. Three hundred seventy-three patients were included in the study. Of the 61 patients with D+C demonstrating hyperplasia, the hysteroscopic impression was hyperplasia in 32 (52%). Of the 10 patients with D+C demonstrating carcinoma, the hysteroscopic impression was hyperplasia in 8 (80%) and carcinoma in 2 (20%). Two additional cases of carcinoma were diagnosed within 6 months of hysteroscopy/D+C, and both had been missed on both hysteroscopy and D+C. Of 204 patients with a normal hysteroscopic impression, 23 (11%) had hyperplasia on D+C. Conclusions. Hysteroscopy did not improve upon the sensitivity of D+C in the detection of endometrial hyperplasia or carcinoma.

Invasive cervical cancer is becoming a rare entity in the United States today. According to National Cancer Institute data, there were 15,800 new cases and 4800 deaths from cervical cancer in 1995 [1]. This is in stark contrast to the statistics from developing nations, where cervical cancer is still the most common malignancy in women. The relatively low incidence of cervical cancer in developed nations is largely attributable to the effectiveness of screening programs [2–5]. Cervical cancer screening today is synonymous with cervical cytologic, or Pap smear, screening. This method was developed to detect precursor lesions, known as cervical intraepithelial neoplasia or dysplasia, as well as early invasive cervical cancer, for which treatments are highly effective.