Efficacy, Immunogenicity, and Safety of a 9-Valent Human Papillomavirus Vaccine: Subgroup Analysis of Participants From Asian Countries.

Background: A 9-valent human papillomavirus-6/11/16/18/31/33/45/52/58 (9vHPV) vaccine extends coverage to 5 next most common oncogenic types (31/33/45/52/58) in cervical cancer versus quadrivalent HPV (qHPV) vaccine. We describe efficacy, immunogenicity, and safety in Asian participants (India, Hong Kong, South Korea, Japan, Taiwan, and Thailand) from 2 international studies: a randomized, double-blinded, qHPV vaccine-controlled efficacy study (young women aged 16-26 years; NCT00543543; Study 001); and an immunogenicity study (girls and boys aged 9-15 years; NCT00943722; Study 002). Methods: Participants (N = 2519) were vaccinated at day 1 and months 2 and 6. Gynecological samples (Study 001 only) and serum were collected for HPV DNA and antibody assessments, respectively. Injection-site and systemic adverse events (AEs) were monitored. Data were analyzed by country and vaccination group. Results: 9vHPV vaccine prevented HPV-31/33/45/52/58-related persistent infection with 90.4%-100% efficacy across included countries. At month 7, ≥97.9% of participants seroconverted for each HPV type. Injection-site AEs occurred in 77.7%-83.1% and 81.9%-87.5% of qHPV and 9vHPV vaccine recipients in Study 001, respectively, and 62.4%-85.7% of girls/boys in Study 002; most were mild to moderate. Conclusions: The 9vHPV vaccine is efficacious, immunogenic, and well tolerated in Asian participants. Data support 9vHPV vaccination programs in Asia. Clinical Trials Registration: NCT00543543; NCT00943722.

Clinical and economic impact of school-based nonavalent human papillomavirus vaccine on women in Singapore: a transmission dynamic mathematical model analysis.

OBJECTIVE: To examine the epidemiological and economic impact of a nine-valent (nonavalent) human papillomavirus (HPV) 6/11/16/18/31/33/45/52/58 vaccine programme for young teenagers in Singapore. DESIGN: Mathematical modelling. SETTING: Pharmaco-economic simulation projection. POPULATION: Singapore demography. METHODS: Clinical, epidemiological and financial data from Singapore were used in a validated HPV transmission dynamic mathematical model to analyse the impact of nonavalent HPV vaccination over quadrivalent and bivalent vaccines in a school-based 2-dose vaccination for 11- to 12-year-old girls in the country. The model assumed routine cytology screening in the current rate (50%) and vaccine coverage rate of 80%. MAIN OUTCOME MEASURES: Changes over a 100-year time period in the incidence and mortality rates of cervical cancer, case load of genital warts, and incremental cost-effectiveness ratio (ICER). RESULTS: Compared with bivalent and quadrivalent HPV vaccination programmes, nonavalent HPV universal vaccination resulted in an additional reduction of HPV31/33/45/52/58 related CIN1 of 40.5%, CIN 2/3 of 35.4%, cervical cancer of 23.5%, and cervical cancer mortality of 20.2%. Compared with bivalent HPV vaccination, there was an additional reduction in HPV-6/11 related CIN1 of 75.7%, and genital warts of 78.9% in women and 73.4% in men. Over the 100 years, after applying a discount of 3%, disease management cost will be reduced by 32.5% (versus bivalent) and 7.5% (versus quadrivalent). The incremental cost-effectiveness ratio (ICER) per quality-adjusted life-year gained was SGD 929 compared with bivalent vaccination and SGD 9864 compared with quadrivalent vaccination. CONCLUSION: Universal two-dose nonavalent HPV vaccination for 11- to 12-year-old adolescent women is very cost-effective in Singapore. TWEETABLE ABSTRACT: Nonavalent HPV vaccination of 11- to 12-year-old girls is cost-effective in Singapore.

No effect of femoral offset on bone implant micromotion in an experimental model.

BACKGROUND: In total hip replacement (THR), the femoral offset (FO) is assessed preoperatively, and the surgeon must determine whether to restore, increase, or decrease the FO based on experience and the patient's clinical history. The FO is known to influence the abductor muscle strength, range of motion (ROM), gait, and hip pain after THR; however, the true effect of FO on bone implant micromotion is unclear. Therefore, we investigated to assess: (1) the muscle loading response during gait, (2) whether FO affects bone implant micromotion during gait. HYPOTHESIS: A variation of ±10mm from the anatomical FO affects the muscle loading forces. MATERIALS AND METHODS: We modified a personalized musculoskeletal model of the lower extremity to determine the 3-dimensional contact forces at the hip joint in the presence of a stem with varying offsets during a gait cycle. A detailed finite element (FE) model was then constructed for increased, restored, and decreased FOs. The maximum load obtained during normal walking gait from the musculoskeletal model was applied to the respective FE models, and the resultant stem-bone micromotion and stress distribution were computed. RESULTS: Increasing the FO to +10mm decreased the peak force generated by the abductor muscles during the cycle by 15.0% and decreasing the FO to -10mm increased the von Mises stress distribution at the distal bone by 77.5% (P<0.05). A variation of the offset within 10mm of the anatomical offset showed no significant differences in micromotion (P>0.05) and peak stresses (P>0.05). DISCUSSION: Coupling the musculoskeletal model of the gait cycle with FE analysis provides a realistic model to understand how FO affects bone implant micromotion. We found that there was no effect of FO on bone implant micromotion; thus, a surgeon does not need to evaluate the implications of FO on micromotion and can determine a FO that best decreases the work load of abductor muscles, increases ROM, and reduces hip pain. LEVEL OF EVIDENCE: IV, biomechanical study.

Clamp for coronary artery operations.

Aortocoronary bypass grafting is an accepted procedure for ischemic heart disease. Proper visualization of the coronary artery is mandatory for good surgical anastomosis. This is essential when a coronary operation is performed without cardioplegia or in surgical procedures without bypass support. For better visualization of a coronary artery, we are presenting a coronary artery clamp. We have used this clamp in minimally invasive coronary artery operations to achieve a bloodless field.

Improved gas chromatography/mass spectrometry analysis of barbiturates in urine using centrifuge-based solid-phase extraction, methylation, with d5-pentobarbital as internal standard.

Effective solid-phase extraction, derivatization, and GC/MS procedures are developed for the simultaneous determinations of butalbital, amobarbital, pentobarbital, and secobarbital, using a deuterated pentobarbital (d5-pentobarbital) as the internal standard. Buffered (pH 7) urine samples were extracted with Bond Elute Certify II cartridge. Iodomethane/tetramethylammonium hydroxide in dimethylsulfoxide was used for methylation, while a HP 5970 MSD equipped with a 13 m J & W DB-5 column (5% phenyl polysiloxane phase) and the Thru-Put Target software package were used for GC/MS analysis and data processing. This protocol was found to be superior, in both chromatographic performance characteristics and quantitation results, over a liquid-liquid extraction procedure without derivatization using hexobarbital as the internal standard. Extraction recoveries observed from control samples containing four barbiturates range from 80% to 90%. Good one-point calibration data are obtained for all four barbiturates in the 50 to 3200 ng/mL range. Interestingly, the one-point calibration data for pentobarbital are inferior to the other three barbiturates--due to interference from the internal standard (d5-pentobarbital). The calibration data of pentobarbital are best described by a hyperbolic curve regression model. Precision data (% CV) for GC/MS analysis, over-all procedure, and day-to-day performance are approximately 2.0%, 6.0%, and 8.0%, respectively. With the use of a 2 mL sample size, the attainable detection limit is approximately 20 ng/mL.

Simultaneous analysis of amphetamine, methamphetamine, and 3, 4-methylenedioxymethamphetamine (MDMA) in urine samples by solid-phase extraction, derivatization, and gas chromatography/mass spectrometry.

A rapid and effective solid-phase extraction procedure using Bond Elute Certify bonded silica sorbent cartridges was adopted to extract amphetamine, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy) from urine samples. The extract was derivatized with trichloroacetic anhydride prior to gas chromatography/mass spectrometry (GC/MS) analysis with selected ion monitoring of the following ions: 190, 91, 188; 204, 91, 202; 162, 135, 202; 194, 123; and 211, 209 for the derivatized amphetamine, methamphetamine, MDMA, d5-amphetamine, and d9-methamphetamine, respectively. The first of the ions listed for each compound was used for quantitation. The compound d5-amphetamine was used as the internal standard for amphetamine, and d9-methamphetamine was used for methamphetamine and MDMA. Results showed a higher than 65% recovery and a reproducibility with less than a 5% coefficient of variation. When a sample size of 2 mL was used, the lowest detectable concentration was about 50 ng/mL, and a near-perfect fit can be obtained (within the 250 to 4000-ng/mL concentration range studied) using a second-order polynomial model.

Enzymatic digestion, solid-phase extraction, and gas chromatography/mass spectrometry of derivatized intact oxazepam in urine.

Enzymatic digestion with beta-glucuronidase (EC 3.2.1.31) was used to release intact oxazepam from urine samples containing the d5-analog internal standard. The resulting specimens were extracted with Du Pont PREP Type W cartridge (processed by a PREP Automated Sample Processor), Bond Elut Certify, and J.T. Baker "spe" columns for comparison of the columns' extraction recovery and overall effectiveness. Methyl iodide/tetrahexylammonium hydrogen sulfate and N,O-bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (10 g/L) were used for the methylation and trimethylsilylation studies. We used a Hewlett-Packard HP 5790 mass-selective detector equipped with a 13-m J & W DB-5 column (5% phenyl polysiloxane phase) for gas chromatography/mass spectroscopy (GC/MS) analysis and the Thru-Put Target software package for data processing. After several exploratory experiments, we adopted the Du Pont PREP system methylation procedure because of its effective recovery, the superior stability of the derivatization product, the possibility of incorporating a clean-up step, and the potential for high throughput. The extraction recovery from a set of control samples was 87%. Coefficients of variation obtained for six replicates of GC/MS analysis and for the overall procedure were 1% and 3%, respectively. Excellent linearity was established in the 50-8000 micrograms/L concentration range studied. With the use of 3-mL samples, a 20-microL final reconstitution volume, oxazepam at 50 micrograms/L was easily detected under the adopted operation conditions.

Simultaneous gas chromatography/mass spectrometry assay of methadone and 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in urine.

An efficient extraction and gas chromatography/mass spectrometry (GC/MS) procedure has been developed for the simultaneous determination of methadone and 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine in urine samples. The merits of this procedure include (1) effective high-volume sample processing; (2) excellent gas chromatography characteristics; (3) high precision for quantitative methadone determination--1.0% coefficient of variation (CV) for GC/MS injection replicates and 1.2% for extraction replicates; (4) excellent linearity within the range (0 to 1200 ng/mL) studied; and (5) adequate detection limits (50 ng/mL) for most practical purposes. The detection limit for methadone may be improved 40-fold by using a different internal standard.

Correlations on radioimmunoassay, fluorescence polarization immunoassay, and enzyme immunoassay of cannabis metabolites with gas chromatography/mass spectrometry analysis of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid in urine specimens.

Results obtained from three commercial immunoassay kits, Abuscreen, TDx, and EMIT, commonly used for the initial test of urine cannabinoids (and metabolites) were correlated with the 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (9-THC-COOH) concentration as determined by GC/MS. Correlation coefficients obtained based on 26 (out of 1359 total sample population) highly relevant samples, are 0.601 and 0.438 for Abuscreen and TDx. Correlation coefficients obtained from a parallel study on a different set of 47 (out of 5070 total sample population) highly relevant specimens are 0.658 and 0.575 for Abuscreen and Emit. The immunoassay concentration levels, that correspond to the commonly used 15 ng/ml GC/MS cutoff value for 9-THC-COOH, as calculated from the regression equations are 82 ng/ml and 75 ng/ml for TDx and EMIT and 120 ng/ml and 72 ng/ml for Abuscreen manufactured at two different time periods. The difference of these calculated corresponding concentrations provides quantitative evidence of the reagent specificity differences.

In vitro effect of acetaldehyde on cell-mediated cytotoxicity by murine spleen cells.

The effects of acetaldehyde in vitro on the lytic capacity of murine spleen cells have been evaluated in three systems: antibody-dependent cell-mediated cytotoxicity (ADCC), natural killer (NK) activity, and alloimmune cytotoxic T lymphocyte (CTL) activity. Acetaldehyde had a biphasic effect on ADCC. Concentrations less than 1 mM acetaldehyde potentiated ADCC. Concentrations greater than 1 mM produced a progressive decrease in lysis. The inhibitory effects were at the effector cell level and were partially irreversible. Preincubation experiments showed that inhibition of ADCC was both concentration and time-dependent. Preincubation of the spleen cells for short periods of time produced potentiated lysis by concentrations of acetaldehyde up to 10 mM. However, potentiation of lysis in preincubation and short term (4h) lytic assay experiments was more variable than longer term (18h) experiments in which the acetaldehyde was not removed by washing. NK activity and alloimmune CTL-mediated lysis were also inhibited by acetaldehyde. Concentrations of acetaldehyde up to 20 mM did not significantly decrease lymphocyte viability as determined by trypan blue exclusion. Acetaldehyde was lost from the reaction mixtures by first order kinetics with a rate constant of 0.5/hr. Thus, the final concentrations were 64-99.99% lower than the starting amounts.

In vitro effects of ethanol and acetaldehyde on lymphoid cells as determined by 31P NMR spectroscopy.

We have used NMR spectroscopy to test the direct in vitro effects of ethanol and its metabolite acetaldehyde on a human leukemic T cell line (Molt-4) and normal murine spleen cells. The metabolic status of phosphate metabolites (phosphomonoesters including sugar phosphate, inorganic phosphate and ATP's) in cell suspension was monitored by 31P NMR spectroscopy. Spectral changes were observed in the intensity of inorganic phosphate and ATP. Addition of high concentrations of ethanol (400-1600 mM) to Molt-4 cells resulted in very little spectral change. However, the addition of 40 microM acetaldehyde resulted in substantially increased inorganic phosphate (Pi) signals, and decreased phosphomonoesters and ATP signals. Similar changes, but to a lesser degree, were observed with normal mouse spleen cells.

Effects of retinoids on human thymus-dependent and thymus-independent mitogenesis.

The effects of all-trans-retinoic acid (RA), 13-cis-retinoic acid, and N-(4-hydroxyphenyl)retinamide on the mitogenic responses of various populations of human lymphocytes have been evaluated. Superoptimal concentrations of mitogens allowed the greatest RA-induced potentiation of lymphocyte proliferation. All three retinoids at concentrations as low as 5 x 10(-14)M significantly potentiated the proliferation of adenoidal and tonsillar lymphocytes stimulated by pokeweed mitogen (PWM). However, the responses of adenoidal and tonsillar lymphocytes to Staphylococcus aureus Cowan strain A were not potentiated by retinoids. Retinoids also caused significant potentiation of proliferation of PWM-stimulated peripheral blood lymphocytes (PBL). However, endpoint concentrations of retinoids required to significantly potentiate PBL proliferative responses to PWM were much higher than required for potentiation of adenoidal or tonsillar lymphocytes. PBL responses to concanavalin A (Con A) were significantly potentiated by retinoid concentrations as low as 10(-8) to 10(-10) M. Retinoid-potentiated responses were also observed wi Con A-stimulated thymocytes, but the endpoint concentrations required for significant potentiation were 10-fold higher than required to potentiate PBL responses to Con A. These data indicate that the sensitivity of lymphocytes to the retinoid-mediated potentiation of mitogenesis depends on the lymphoid compartment from which the cells are obtained. Tonsillar and adenoidal lymphocytes were the most responsive of the lymphocytes tested to the retinoid-induced potentiation of PWM responses. In addition, retinoids appear to selectively potentiate T cell-dependent proliferative activity.

Effects of retinoids on macrophage function and IL-1 activity.

The effects of three retinoids, all-trans-retinoic acid (RA), 13-cis-retinoic acid (cRA), and N-(4-hydroxyphenyl) retinamide (4-HPR), on macrophage function were evaluated. In vitro, RA, cRA, and 4-HPR caused a greater than twofold increase in phagocytosis of IgG-sensitized bovine erythrocytes (IgG-ORBC) by a mouse macrophage cell line (RAW). Significant increases in phagocytosis were produced by retinoid concentrations as low as 2 x 10(-10) M. RA also significantly increased phagocytosis of IgG-sensitized ORBC by BALB/c peritoneal macrophages in vitro. The ability of RAW macrophages to bind IgG-ORBC was significantly increased by 10(-6) to 10(-14) M RA. The potentiation of mitogenic responses of spleen cells to Con A and PWM by RA was relatively independent of macrophage function, i.e., splenocytes that were macrophage-depleted were responsive to the potentiating effects of RA. The effects of retinoids on T-cell-dependent B-cell mitogenesis induced by PWM appeared not to be dependent on their previously reported capacity to alter prostaglandin synthesis. Treatment of spleen cells with 10(-6) M indomethacin did not abolish the potentiating effects of RA. However, RA in a dose-dependent fashion increased IL-1 activity at the level of the target T-cell. The greatest potentiation of IL-1 activity was at 10(-8) M RA. These results show that retinoids can modulate macrophage function at two different levels: potentiation of phagocytosis and potentiation of IL-1 activity at the level of the T-cell.

Monoclonal IgM antibodies that inhibit primary Moloney murine sarcoma growth.

Monoclonal IgM antibodies with specificity for Moloney murine sarcoma virus (M-MuSV)-Moloney murine leukemia virus (M-MuLV) from two hybridoma clones have been isolated and characterized. The monoclonal antibodies have specificity for a cytoplasmic and cell surface Friend-Moloney-Rauscher group-specific antigen. Immunoelectron microscopy revealed antibody binding to the surface of virus-expressing cells but not to the budding virus particles. Treatment of M-MuSV-injected mice with monoclonal IgM anti-M-MuSV significantly inhibited tumor growth compared to virus-inoculated animals receiving either saline or MOPC 104E. Nude mice exhibited delayed tumor induction following treatment with the monoclonal antibodies but ultimately died from tumor growth. Virus-injected euthymic mice that were treated with monoclonal IgM anti-M-MuSV generated a potentiated spleen cell-mediated cytotoxicity against Moloney sarcoma cells compared to virus-infected treated with saline. This potentiation of cytotoxicity remained after trypsinization of the spleen cells and thus was probably not due to passively adsorbed monoclonal antibody. The antibodies alone or in the presence of complement did not neutralize M-MuLV. The IgM antibodies induced specific tumor cell cytotoxicity in vitro mediated by complement spleen cells, lymph node cells, or thymus cells. In conclusion, two monoclonal IgM anti-M-MuSV antibodies that bind to the tumor cell surface did not neutralize virus can inhibit primary M-MuSV-induced tumor growth in vivo. The regression event appeared to involve heterogeneous mechanisms. Complete regression remained thymus dependent even with passive antibody therapy, but significant tumor growth inhibition was produced independent of T-cells. In vitro these IgM antibodies induced complement and cell-mediated cytotoxicity.

In vitro effect of ethanol on cell-mediated cytotoxicity by murine spleen cells.

The effects of ethanol on murine spleen cell-mediated lysis have been studied. Concentrations of 5.5-176 mM ethanol produced progressive inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC). Binding of spleen cells to antibody-sensitized target cells was not inhibited by comparable concentrations of ethanol. Kinetic analysis revealed decreased rates of lysis with increasing concentrations of ethanol. Changes of effector to target cell ratios revealed an inhibition of maximum lysis and decreased lytic efficiency in the presence of 88 mM ethanol. Preincubation experiments showed the inhibitory effect of ethanol to be reversible. Macrophage-depleted spleen cells appeared to be as susceptible to inhibition by ethanol as unfractionated spleen cells. Ethanol also inhibited natural killer and alloimmune cytotoxic T cell activity. The ADCC data were analysed by using a mathematical model which incorporates the kinetics of lysis, dose-response relationships, heterogeneity of the lytic effectors, reversibility of inhibition and ethanol loss during incubation. An inhibition constant (KI) of 373 mM-2 when two ethanol molecules interact with the site of inhibition was calculated. 50% inhibition of lysis is produced by 52 mM (0.24%) ethanol. The results are consistent with a model which assumes that lysis is due to a critical number of interactions which ultimately trigger the lytic event. Alcohol interferes with lysis by reacting with sites which are required for triggering the lytic event. Although the molecular details of the mechanism of inhibition are as yet undefined, we infer that ethanol inhibits ADCC at the programming for lysis or the lethal hit stages.

Immune response to polyoma tumor cells in mice--III. Stimulation of tumor cell growth in vitro by spleen cells from immunized animals.

We have evaluated the stimulation of target cell growth in vitro by spleen cells from mice which were immunized with polyoma-transformed cells and other tumor and non-tumor antigens. Stimulation was particularly seen under conditions of immunization that were suboptimal for the production of specific cytotoxicity. Significant stimulation of polyoma target cell growth was observed by lymphocytes from mice immunized against 10(5) Py 4198 tumor cells. This stimulation of target cell growth was not confined to polyoma-transformed cells only. Cells transformed by SV40, H-MuSV and non-transformed cells like 3T3 and embryo fibroblasts were also stimulated. Immunization of mice with syngeneic embryo fibroblasts also resulted in stimulation of tumor cell growth by the spleen cells from the immunized mice. However, the growth stimulation was less consistent and did not occur in all target cells tested. The specificity of immunostimulation was further studied with the Moloney sarcoma virus (M-MuSV) system; an antigenically distinct tumor system. Spleen cells from M-MuSV tumor-bearing mice stimulated cell growth in vitro not only against MuSV-transformed cells but also with SV40-transformed and polyoma-transformed cells as targets. Significant stimulation of target cell growth was also observed by spleen cells from mice that were immunized against 'non-pertinent' antigens, e.g. sheep red blood cells and allogeneic (C57B 1/6) spleen cells.

Murine T cells that lyse antibody-sensitized target cells. III. Contribution of Thy 1-Bearing cells to the lytic activity of normal spleen.

Murine spleen cells were analysed for their capacity to lyse 51Cr-labelled antibody-sensitized erythrocytes and a human lymphoma cell line. Incubation of spleen cells with iron powder followed by removal of iron-containing cells with a magnet significantly decreased the lytic capacity of the remaining cells against erythrocyte target cells. However, substantial cytotoxicity remained in the relatively phagocyte-depleted population. Both antibody-sensitized erythrocytes and tumour cells were lysed by phagocyte-depleted effector cells. However, more spleen cells were required to lyse nucleated target cells than were required to produce comparable lysis of the erythrocytes. Such phagocyte-depleted spleen cells were subsequently treated with three different antisera containing specificities for thymus-dependent antigens and a monoclonal IgM anti-Thy 1.2 in the presence of complement. The remaining viable cells were recovered and tested as effector cells. All four reagents in the presence of complement caused an inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC) that was proportional to the percentage of T cells eliminated. However, the antisera also inhibited ADCC in the absence of complement, even when the cells were trypsinized following the antiserum treatment to remove attached antibodies. On the other hand, treatment of spleen cells with the monoclonal IgM anti-Thy 1.2 followed by trypsin treatment did not inhibit ADCC unless complement was added. Thus, with the latter reagent, decreased ADCC could be ascribed to elimination of T cells and not immune complex inhibition. Cells bearing Thy 1.2 accounted for approximately half of the lytic activity of phagocyte-depleted spleen cells against antibody-sensitized target cells.