Biomedical subjects

Philip E Castle

Publications and source records attributed to Philip E Castle.

64 records · Page 4Linked to original sources

PubMed2002

Viral load of human papillomavirus and risk of CIN3 or cervical cancer.

Carcinogenic human papillomaviruses (HPV) are thought to be necessary for development of cervical cancer. We assessed whether higher viral loads of such viruses predicted future risk of CIN3 or cancer (CIN3+) in a cohort of 20810 women followed up for 10 years with cytological screening. We measured the viral load for 13 types of carcinogenic HPV (relative light units normalised to 1 pg/mL HPV 16 positive controls [RLU/PC]) using Hybrid Capture 2 testing of cervicovaginal lavages obtained at enrolment. Results were stratified into four groups (RLU/PC 1 to <10, 10 to <100, 100 to <1000, > or = 1000). Although presence of HPV strongly increased risk of CIN3+, high viral load did not further predict risk of CIN3+.

Adolescent↗

PubMed2002

Comparisons of HPV DNA detection by MY09/11 PCR methods.

Two modifications to the original L1 consensus primer human papillomavirus (HPV) PCR method, MY09-MY011, using AmpliTaq DNA polymerase (MY-Taq), were evaluated for HPV DNA detection on clinical specimens from a cohort study of cervical cancer in Costa Rica. First, HPV DNA testing of 2978 clinical specimens by MY09-MY011 primer set, using AmpliTaq Gold DNA polymerase (MY-Gold) were compared with MY-Taq testing. There was 86.8% total agreement (kappa = 0.72, 95%CI = 0.70-75) and 69.6% agreement among positives between MY-Gold and MY-Taq. MY-Gold detected 38% more HPV infections (P < 0.0001) and 45% more cancer-associated (high-risk) HPV types (P < 0.0001) than MY-Taq, including 12 of the 13 high-risk HPV types. Analyses of discordant results using cytologic diagnoses and detection of HPV DNA by the Hybrid Capture 2 Test suggested that MY-Gold preferentially detected DNA positive specimens with lower HPV viral loads compared with MY-Taq. In a separate analysis, PGMY09-PGMY11 (PGMY-Gold), a redesigned MY09/11 primer set, was compared with MY-Gold for HPV DNA detection (n = 439). There was very good agreement between the two methods (kappa = 0.83; 95%CI = 0.77-0.88) and surprisingly no significant differences in HPV detection (P = 0.41). In conclusion, we found MY-Gold to be a more sensitive assay for the detection of HPV DNA than MY-Taq. Our data also suggest that studies reporting HPV DNA detection by PCR need to report the type of polymerase used, as well as other assay specifics, and underscore the need for worldwide standards of testing.

Cervix Uteri↗

PubMed2002

Cervical concentrations of interleukin-10 and interleukin-12 do not correlate with plasma levels.

Human papillomavirus (HPV) infects the transformation zone of the cervix and is the primary cause of cervical cancer. The infection is localized to the cervix and mucosal immunity is likely to be an important determinant for viral clearance. Previous studies of immunity to HPV have measured immune markers in the blood, but the relationship of systemic immunity to cervical immunity is poorly understood. In this study of 70 women enrolled in the ASCUS-LSIL Triage Study (ALTS), a clinical trial for management of low-grade cytologic abnormalities of the cervix, we collected paired plasma and cervical secretions to investigate the relationship between cervical concentrations of interleukin-10 (IL-10) and interleukin-12 (IL-12) and plasma levels. Neither IL-10 (p = 0.11), or IL-12 (p = -0.04) nor the ratio of IL-12 to IL-10 (p = 0.06) were correlated between blood and cervical secretions. Except for weak correlations of IL-10 among nonsmokers (p = 0.35. P = 0.019) and those in day 18-27 of their menstrual cycle (p = 0.51, P = 0.015), this lack of correlation persisted in all subgroups defined by genital inflammation or infection, current oral contraceptive use, heme contamination and volume of collected secretions, HPV16 seropositivity, and repeat HPV infection and/or cytologic abnormalities. The lack of correlation and high concentrations in cervical secretions indicate that the cervical IL-10 and IL-12 concentrations exceed what could be expected from blood as a principle source of IL-10 and IL-12 and suggest that cytokine concentrations in cervical secretions are predominantly the result of local cytokine production.

Adult↗

PubMed2002

Subcellular distribution of ZP1, ZP2, and ZP3 glycoproteins during folliculogenesis and demonstration of their topographical disposition within the zona matrix of mouse ovarian oocytes.

The zona pellucida (ZP) is an extracellular coat synthesized and secreted by the oocyte during follicular development and surrounding the plasma membrane of mammalian eggs. To date, the mechanism of synthesis and secretion, mode of assembly, and intracellular trafficking of the ZP glycoproteins have not been fully elucidated. Using antibodies against mouse ZP1, ZP2, and ZP3 in conjunction with the protein A-gold technique, we have shown an association of immunolabeling with the Golgi apparatus, secretory granules, and a complex structure called vesicular aggregate, respectively, in mouse ovarian follicles. In contrast, the neighboring granulosa cells were not reactive to any of the three antibodies used. Immunolabeling of ZP1, ZP2, and ZP3 was detected throughout the entire thickness of the ZP, irrespective of the developmental stage of ovarian follicles. Double and triple immunolocalization studies, using antibodies tagged directly to different sizes of gold particles, revealed an asymmetric spatial distribution of the three ZP glycoproteins in the zona matrix at various stages of follicular development. All three glycoproteins were specifically localized over small patches of darkly stained flocculent substance dispersed throughout the zona matrix. Very often, ZP1, ZP2, and ZP3 were found in close association. These results confirm findings from previous studies demonstrating that ovarian oocytes and not granulosa cells are the only source for mouse ZP glycoproteins. In addition, results from our morphological and immunocytochemical experiments suggest that the vesicular aggregates in the ooplasm are likely to serve as an intermediary in the synthesis and secretion of ZP glycoproteins. The stoichiometric disposition of ZP1, ZP2, and ZP3 in the zona matrix as revealed by double and triple immunolocalization studies provide further insight into some of the unanswered questions pertinent to the current model of mouse ZP structure proposed by the Wassarman group.

Animals↗

PubMed2002

Sexual behavior, human papillomavirus type 16 (HPV 16) infection, and HPV 16 seropositivity.

BACKGROUND: Sexual behaviors have been linked to seropositivity for human papillomavirus (HPV) but not with the magnitude of the seroreactivity. GOALS: The objective of this analysis was to examine the association of sexual behavior, cervical HPV 16 DNA positivity at enrollment (past) and at diagnosis (current), and other potential determinants with the likelihood and magnitude of HPV 16 seropositivity at diagnosis. STUDY DESIGN: With use of stored specimens from an incidence case-control study at Kaiser Permanente (Portland, OR), women were tested for seroreactivity to HPV 16 by enzyme-linked immunosorbent assay with virus-like particles at diagnosis and were tested for past and concurrent cervical HPV 16 DNA positivity with MY09/MY11 L1 consensus primer PCR. Questionnaire data were used to ascertain past sexual behavior. RESULTS: Increased lifetime number of sex partners (P(Trend) < 0.001), past HPV 16 DNA positivity (odds ratio = 6.9; 95% confidence interval = 1.5-31), and a current cytologic diagnosis (P(Trend) < 0.03) were independently associated with HPV 16 seropositivity. Among the seropositive, only lifetime number of sex partners (P(Trend) < 0.001) and past HPV 16 DNA positivity (P = 0.003) were independently associated with mean signal strength (optical density) in an age-adjusted analysis. Women negative for past and concurrent HPV 16 DNA had a significant trend of increasing optical densities associated with greater numbers of lifetime partners (P(Trend) < 0.001). Conversely, the mean signal strength for those women who were ever HPV 16 DNA-positive during the study did not depend on lifetime numbers of sex partners (P(Trend) = 0.36). CONCLUSIONS: HPV 16 seropositivity is a surrogate for past HPV 16 infection. Circulating levels of antibodies to HPV 16 may reflect recent HPV 16 infection or the frequency of past HPV 16 infection.

Adult↗

PubMed2002

Results of human papillomavirus DNA testing with the hybrid capture 2 assay are reproducible.

Reproducibility of the Hybrid Capture 2 Test (HC 2) for human papillomavirus (HPV) DNA detection was evaluated by assaying frozen cervical specimens in 1997 and again in 2001 from 1,775 women with normal cervical cytology. Using a cutoff point of 1.0 pg of HPV DNA/ml between a negative and a positive test result, the result of the kappa test for agreement was 0.72 (a kappa value of >0.60 is considered good agreement). Using cutoff points of 1.0 and 10.0 pg/ml between negative and low positive and between low positive and high positive, respectively, the kappa was 0.68 and the linear-weighted kappa was 0.76. The results of this study indicate that HC 2 testing is reproducible even among cytologically normal women with low test values.

Cervix Uteri↗

PubMed2002

Could multiple low-affinity bonds mediate primary sperm-zona pellucida binding?

The current model for primary sperm binding to the zona pellucida is that a cell-surface sperm protein binds with high affinity in an order-specific manner to one of the zona pellucida proteins, ZP3. However, the molecular details of primary sperm-zona pellucida binding remain elusive. A possible revised model is that multiple low-affinity bonds between spermatozoa and the zona pellucida may be sufficient for primary binding. The avidity of several low-affinity bonds can exceed that of a single high-affinity bond, which is sufficient to tether a motile spermatozoon. A mechanism involving multiple low-affinity bonds could account for some of the difficulties in elucidating primary sperm-zona pellucida interactions.

Animals↗

PubMed2002

Restricted cross-reactivity of hybrid capture 2 with nononcogenic human papillomavirus types.

Hybrid Capture 2 Test using probe B (HC2-B) is a clinical test for the detection of 13 human papillomavirus (HPV) types associated with cervical cancer (oncogenic types), but the potential clinical significance of HC2-B cross-reactivity with untargeted (nononcogenic) HPV types has not been fully evaluated. Thus, HC2-B test results on 954 clinical cervical specimens from a population-based natural history study of HPV in Costa Rica were compared with the data from testing of the same specimens twice by HPV type-specific MY09/MY11 L1 consensus primer PCR. Specimens positive by PCR for single HPV types not targeted by HC2-B were used for determining type-specific cross-reactivity. Effects of cross-reactivity on clinical performance were estimated by calculating sensitivity and specificity with and without cross-reactivity for the detection of high-grade cervical lesions. HC2-B tested positive for single infections by untargeted (cross-reactive) types 11, 53, 61, 66, 67, 70, 71, and 81. Cross-reactivity was strongly associated with PCR signal strength (P(Trend) = 0.0001) and cervical abnormalities (P = 0.0002, Pearson chi(2)). We estimated that HC2-B cross-reactivity resulted in minor changes in screening performance. Clinical sensitivity increased from 84.3% to 87.9%, clinical specificity decreased from 89.6% to 88.1%, and referral rates increased from 11.7% to 13.2% for detection of >or=cervical intraepithelial neoplasia grade 2. The clinical effect of cross-reactivity varied by cytologic interpretation. Among women with normal cytologic interpretations, cross-reactivity significantly improved the accuracy of identifying cytologically nonevident histology of >or=cervical intraepithelial neoplasia grade 2 because of increased sensitivity with maintained specificity. However, among women with equivocal or mildly abnormal cytologic interpretations, cross-reactivity decreased the accuracy of HPV testing because of substantial decreases in specificity. In summary, cross-reactivity with nononcogenic HPV types had little effect on the overall clinical performance of HC2-B as a general screening test, but reduction of cross-reactivity might improve the performance of HPV testing for triage of equivocal or mildly abnormal cytologic interpretations.

Adult↗

PubMed2002

Simultaneous measurement of several cytokines using small volumes of biospecimens.

The role of host immunity in the development of virus-induced cancers has been difficult to elucidate, in part because of our inability to effectively measure multiple immune parameters using available amounts of biological material. The objective of the present study was to validate the use of a newly developed multiplex assay, the LINCOplex assay, for the simultaneous measurement of multiple cytokines [interleukin/(IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IFN-gamma, and tumor necrosis factor-alpha]. Supernatants obtained from peripheral blood mononuclear cell cultures stimulated with various different mitogens and antigens (including phytohemagglutinin, influenza, tetanus, HPV16 E6 and E7 peptides, and media alone) were selected for study. In total, 750 specimens obtained from 26 participants were tested in replicate using the 8-plex LINCOplex assay (25 micro l of specimen required per well). Every specimen was included in duplicate in a blinded fashion. For some specimens, multiple 2-fold dilutions of the same specimen were included to evaluate the linearity of results. The availability of independently obtained IL-2 and IFN-gamma results from standard single cytokine (simplex) assays allowed for a direct comparison between the LINCOplex results and those obtained from the simplex assays. Spearman correlation coefficients for continuous results, and exact agreement rates and weighted kappa statistics for quartiled variables, were computed to evaluate intra- and interassay agreement. IL-4 levels were consistently below the detectable level of the assay (3 pg/ml) whereas IL-6 and IL-8 levels were consistently above the highest detectable level of the assay (10,000 pg/ml), and these three cytokines were, therefore, not evaluated further. For the remaining five cytokines, excellent intra-assay reproducibility was observed, with Spearman correlation coefficients consistently above 0.90 for all five cytokines. Exact agreement rates ranging from 77.6-90.3% and weighted kappas ranging from 0.81-0.92 were observed. Similar results were observed when analysis was restricted to supernatants obtained from cultures that had been stimulated with HPV16 peptides and when analysis was restricted to supernatants obtained from cultures containing no antigen or mitogen, suggesting that the LINCOplex assay is applicable under conditions where moderate or weak cytokine responses/levels are expected. Linearity of results was observed when dilutions of a single specimen were blindly tested, with the exception of IL-2 and IL-10, where deviations from linearity were sometimes observed. For IL-2 and IFN-gamma, where results from simplex assays were available for comparison, the LINCOplex assay and the simplex assay results agreed well. Spearman correlation coefficients were 0.86 and 0.93 for IL-2 and IFN-gamma, respectively. Exact agreement and weighted kappa values were 68.5% and 0.72 (95% confidence interval, 0.65-0.79), respectively, for IL-2 and 67.3% and 0.73 (95% confidence interval, 0.67-0.80), respectively, for IFN-gamma. These results indicate the applicability of the LINCOplex assay for the simultaneous measurement of multiple cytokines using small amounts of biological material.

Biological Assay↗

PubMedDate not supplied

Human monoclonal antibody stability and activity at vaginal pH.

Antibodies can be delivered topically to the vagina to protect against pregnancy and sexually transmitted infections, but the acidity of vaginal secretions (pH 3.5-4.5) might inactivate them. To address this question, both experimental and computational methods were used to evaluate the effects of pH on human monoclonal antibody (MAb) stability and activity. To determine the acid-sensitivity of their antigen binding sites, human MAbs against human sperm (H6-3C4) and gp120 of HIV (1511) were tested by ELISA for binding to human sperm and recombinant gp120, respectively, at pH 3.0-7.0, after storing them for 1 or 20 h at the same pH. Binding was unaltered by acidic pH> or =4 even after 20 h, and at pH 3.5 both MAbs retained > or =40% antigen binding activity. A humanized MAb against HSV-2 glycoprotein B expressed both in Chinese hamster ovary (CHO) cells and in soybean cells was incubated for 1 or 24 h at pH 3.5-7.6, brought to neutral pH, and tested for ability to block HSV-2 infection of foreskin fibroblast cells. Loss in blocking activity occurred only when antibodies were incubated at pH 3.5 for 24 h and was independent of the expression cell type. Using empirical structure-based methods, net charge, Z, and electrostatic contributions to free energy, DeltaDeltaG(el), were calculated as a function of pH for 1 human and 8 murine F(ab)s. The calculations indicate that Z changes slowly between pH 5.0 and 9.0 and that DeltaDeltaG(el) is nearly constant between pH 4.0 and 10 for all the F(ab)s and, therefore, human antibodies should remain stable in this pH range. Taken together, our data and empirical calculations suggest that vaginally applied human MAbs are likely to remain stable and active throughout the duration they are likely to reside in the vagina.

Antibodies, Monoclonal↗