Development and acceptability of a video-based vaccine promotion tutorial for obstetric care providers.

A prenatal care provider's recommendation for maternal vaccines is one of the strongest predictors of vaccine acceptance during pregnancy. Aside from basic talking points, few resources exist to help obstetric care providers effectively navigate conversations with vaccine hesitant patients. This paper describes the development and acceptability of "VaxChat," an hour-long, evidence-based video tutorial aimed at improving obstetric care providers' ability to promote maternal vaccines. Between June and November 2017, 62 obstetric care providers registered to receive continuing medical education credit for viewing VaxChat. Of the post-tutorial responses received, over 90% said VaxChat increased their knowledge of what to say to vaccine hesitant patients, increased their confidence in addressing vaccinations with their pregnant patients, and will help them improve their practice culture regarding maternal vaccine promotion. Eighty percent intend to change how they approach vaccine conversations. These data suggest VaxChat may be a welcome complement to existing provider-to-patient talking points.

Improving influenza and Tdap vaccination during pregnancy: A cluster-randomized trial of a multi-component antenatal vaccine promotion package in late influenza season.

BACKGROUND: Evidence-based interventions to improve influenza vaccine coverage among pregnant women are needed, particularly among those who remain unvaccinated late into the influenza season. Improving rates of antenatal tetanus, diphtheria and acellular pertussis (Tdap) vaccination is also needed. PURPOSE: To test the effectiveness of a practice-, provider-, and patient-focused influenza and Tdap vaccine promotion package on improving antenatal influenza and Tdap vaccination in the obstetric setting. METHODS: A cluster-randomized trial among 11 obstetric practices in Georgia was conducted in 2012-2013. Intervention practices adopted the intervention package that included identification of a vaccine champion, provider-to-patient talking points, educational brochures, posters, lapel buttons, and iPads loaded with a patient-centered tutorial. Participants were recruited from December 2012-April 2013 and included 325 unvaccinated pregnant women in Georgia. Random effects regression models were used to evaluate primary and secondary outcomes. RESULTS: Data on antenatal influenza and Tdap vaccine receipt were obtained for 300 (92.3%) and 291 (89.5%) women, respectively. Although antenatal influenza and Tdap vaccination rates were higher in the intervention group than the control group, improvements were not significant (For influenza: risk difference (RD)=3.6%, 95% confidence interval (CI): -4.0%, 11.2%; for Tdap: RD=1.3%, 95% CI: -10.7%, 13.2%). While the majority of intervention package components were positively associated with antenatal vaccine receipt, a provider's recommendation was the factor most strongly associated with actual receipt, regardless of study group or vaccine. CONCLUSIONS: The intervention package did not significantly improve antenatal influenza or Tdap vaccine coverage. More research is needed to determine what motivates women remaining unvaccinated against influenza late into the influenza season to get vaccinated. Future research should quantify the extent to which clinical interventions can bolster a provider's recommendation for vaccination. This study is registered with clinicaltrials.gov, study ID NCT01761799.

The role of complement receptor 3 (CR3) in Neisseria gonorrhoeae infection of human cervical epithelia.

Neisseria gonorrhoeae is an important sexually transmitted pathogen and a major cofactor in HIV-1 infection. This organism uses different mechanisms to infect male and female genital tract epithelia. Receptor-mediated endocytosis of N. gonorrhoeae is the principle mechanism of entry into male urethral epithelial cells. Infection in men leads to a pronounced inflammatory response. In contrast, N. gonorrhoeae infection in women induces ruffling of the cervical epithelia, allowing a macropinocytic mechanism of entry. Infection in women is frequently asymptomatic, suggesting suppression of the inflammatory response. N. gonorrhoeae-induced membrane ruffling and inflammation suppression are consistent with the ability of this bacterium to enter cervical epithelial cells, in vitro and in vivo, by interaction with complement receptor 3 (CR3), a receptor that does not trigger an inflammatory response. This receptor is present on cervical epithelial cells but not on male urogenital tract epithelia. N. gonorrhoeae engagement of CR3 initiates a unique mechanism of bacterial-induced membrane ruffling and internalization. These studies explain why the pathology of N. gonorrhoeae infection differs between males and females. Additionally, the observation that this receptor is present on cervical epithelia may provide insight into the pathogenesis of other sexually transmitted pathogens.

Chlamydia trachomatis infection induces mucosal addressin cell adhesion molecule-1 and vascular cell adhesion molecule-1, providing an immunologic link between the fallopian tube and other mucosal tissues.

The development of a protective vaccine against the sexually transmitted disease caused by Chlamydia trachomatis may prevent complications associated with insidious infection. Vaccination via the vaginal route may not be practical, and other routes should be investigated. To this end, the adhesion molecules induced on the fallopian tube endothelium during infection with C. trachomatis were characterized. Adhesion molecules were identified in fallopian tube biopsy specimens cultured with 5 x 10(6) infection-forming units of C. trachomatis serovar E. Frozen sections were prepared from these tissues and were stained by immunohistochemical techniques. Infection with live, but not UV-inactivated, C. trachomatis induced a significant increase in levels of vascular cell adhesion molecule-1 and the mucosal addressin cell adhesion molecule-1 but not of other adhesion molecules. Therefore, infection with C. trachomatis induces adhesion molecules that are associated with other mucosal tissues and inflammatory sites, which suggests that mucosal routes of immunization may be effective.

The clinical utility of flow cytometry in the study of platelets.

Flow cytometry has emerged in the past few years as an important technology for the study of platelets. It offers the ability to make measurements on platelets with little or no isolation or manipulation. Most flow cytometric platelet studies can be carried out on whole blood, thus eliminating a host of artifacts. In addition, flow cytometric techniques have been developed that allow the measurement of nearly all of the functional capabilities of platelets, such as activation and aggregation and to identify new functions by permitting observation of platelets interacting with leukocytes and measurement of platelet microparticles. Several of these measurements have already reached the stage of clinical utility and others offer considerable promise for practical applications. This review describes each of the flow cytometric techniques used to study platelets and summarizes their current state of clinical utility. Semin Hematol 38:160-168.

Abnormal platelet function and calcium handling in Dahl salt-hypertensive rats.

The effect of dietary salt on platelet function and Ca(2+) homeostasis was studied in Dahl (DS) rats, a genetic model of salt-sensitive hypertension. DS rats were fed a high-salt (DSHS) or a low-salt diet (DSLS) for up to 4 weeks, and the effects of salt loading on systolic blood pressure, platelet P-selectin expression, and platelet Ca(2+) homeostasis were measured. The high-salt diet increased blood pressure and markedly increased the amount of ionomycin (IM)-releasable Ca(2+) in platelet intracellular stores (Ca(2+)/IM). The alteration in Ca(2+) stores was not prevented when the hypertension was prevented by treatment with hydralazine and reserpine. The Ca(2+) store filling during platelet exposure to 1 mmol/L Ca(2+) for 5 minutes and the rate of sarcoplasmic/endoplasmic Ca(2+) ATPase-dependent Ca(45) uptake were higher in DSHS compared with that in DSLS. There was a decrease in thrombin-induced Ca(2+) influx in platelets from DSHS; consistent with this, agonist-induced P-selectin expression was decreased. In DSLS, nitric oxide accelerated reloading of platelet Ca(2+) stores after their emptying by thrombin but failed to do so in DSHS. These results indicate that in DS rats, a high-salt diet increases sarcoplasmic/endoplasmic Ca(2+) ATPase activity and the Ca(2+)/IM but decreases the reuptake of Ca(2+) caused by nitric oxide. Decreases in Ca(2+) influx and platelet P-selectin expression might be explained by changes in intracellular Ca(2+) stores in DSHS rats, which apparently is a heritable response to a high-salt diet.

Recurrent carcinoma in situ of a neovagina.

BACKGROUND: Development of carcinoma in situ in a neovagina is rare. CASE: A case of carcinoma in situ of a neovagina complicated by recurrence after ablative therapy is discussed. Recurrence occurred within 4 months of initial therapy, and a total vaginectomy was performed after the patient declined other therapeutic options. CONCLUSION: Recurrent carcinoma in situ of a neovagina can be successfully treated by surgical excision.

An interlaboratory study of a candidate reference method for platelet counting.

A multinational interlaboratory task force explored the important variables of platelet reference counting and developed a candidate flow cytometric reference method based on the RBC/platelet ratio. A multicenter comparison was performed to determine whether the method met the necessary criteria and was precise enough to be recommended as a new reference method. Each laboratory analyzed serial dilutions of normal specimens, stabilized material, and at least 60 patient specimens with a range of platelet counts from 1 to 400 x 10(3)/microL (1-400 x 10(9)/L). Pooled analysis of the serial dilutions showed that RBC-platelet and RBC-RBC coincidence events became negligible at sufficiently high dilutions (i.e., > 1:1,000). All laboratories demonstrated excellent intra-assay and acceptable interlaboratory precision. Two antibodies (CD61 and CD41) were used for identifying platelets and individually gave acceptable results, but in a minority of samples, staining differences were observed. The optimum method thus uses a double-labeling procedure with a final dilution factor of 1:1,000. The study demonstrated that this method meets the criteria for a reference platelet count.

Neisseria gonorrhoeae elicits membrane ruffling and cytoskeletal rearrangements upon infection of primary human endocervical and ectocervical cells.

Neisseria gonorrhoeae is a strict human pathogen that is, primarily, transmitted by close sexual contact with an infected individual. Gonococcal infection of the male urogenital tract has been well studied in experimental human models and in urethral cell culture systems. Recent studies, using tissue culture cell systems, have suggested a role for the cervical epithelium in gonococcal infection of females; however, the nature of gonococcal infection of the normal uterine cervix remains controversial. To address this enigma, we have developed two primary human cervical epithelial cell systems from surgical biopsies. Gonococcal infection studies and electron microscopy show that N. gonorrhoeae is capable of infecting and invading both the endo- and the ectocervix. Invasion was found to occur primarily in an actin-dependent manner, but it does not appear to require de novo protein synthesis by either the bacterium or the host cervical cell. Membrane ruffles appear to be induced in response to gonococci. Consistent with membrane ruffling, gonococci were found residing within macropinosomes, and a concentrated accumulation of actin-associated proteins was observed to occur in response to gonococcal infection. Electron microscopy of clinically derived cervical biopsies show that lamellipodia formation and cytoskeletal changes, suggestive of membrane ruffles, also occur in the cervical epithelium of women with naturally acquired gonococcal cervicitis. These studies demonstrate the ability of N. gonorrhoeae to infect and invade both the endo- and the ectocervix of the normal uterine cervix. Gonococcal induced ruffling is a novel finding and may be unique to the cervical epithelium.

High levels of platelet inhibition with abciximab despite heightened platelet activation and aggregation during thrombolysis for acute myocardial infarction: results from TIMI (thrombolysis in myocardial infarction) 14.

BACKGROUND: We evaluated platelet activation and aggregation in patients with acute myocardial infarction (AMI) treated with thrombolytic therapy alone or with reduced-dose thrombolysis and concomitant abciximab. METHODS AND RESULTS: The study was performed in 20 control subjects and 51 patients with AMI before and after reperfusion with either alteplase or reteplase or reduced doses of these agents with concomitant abciximab. Platelet activation was assayed by platelet surface expression of P-selectin. Turbidometric platelet aggregation in response to ADP was measured in patients before thrombolytic therapy and 90 minutes and 24 hours after the beginning of thrombolytic therapy. P-selectin expression was greater at baseline in patients than normal control subjects (30.4% versus 9. 8%, P<0.0001) but was identical between the 2 groups after stimulation with ADP (64.4% versus 69.3%, P=0.37). However, at 24 hours, basal P-selectin expression declined in patients (P=0.0025 versus baseline), whereas ADP-stimulated P-selectin expression was lower in patients than in control subjects (48% versus 69%, P=0. 0004). When combined with reduced doses of either alteplase or reteplase, abciximab achieved 91% and 83% inhibition of 5 and 20 micromol/L ADP-induced platelet aggregation, which decreased to 46% and 40%, respectively, at 24 hours. No appreciable difference in the platelet inhibition profile of abciximab was observed between the 2 thrombolytics. CONCLUSIONS: Platelet activation and aggregation are heightened in the setting of thrombolysis for AMI. Despite this enhanced level of platelet activation, abciximab, combined with a reduced-dose thrombolytic, inhibited platelet aggregation similarly to the level reported in elective settings.

Seroreactivity to Chlamydia trachomatis Hsp10 correlates with severity of human genital tract disease.

We have identified the chlamydial heat shock protein Hsp10 as a potential correlate to the immunopathogenic process in women with tubal factor infertility (TFI). The human serologic response to chlamydial Hsp10, Hsp60, and major outer membrane protein (MOMP) was measured by enzyme-linked immunosorbent assay. Three populations of women were studied: uninfected controls (CU), acutely infected (AI) women, and women with TFI. Sera from women in the AI and TFI groups both recognized Hsp10 more frequently and at a higher overall level than sera from healthy uninfected controls. Moreover, the infertile women had significantly greater Hsp10 seroreactivity than acutely infected women, indicating a concomitant increase of Hsp10 recognition in populations with increasing levels of disease severity. Hsp60 reactivity showed a similar correlation in these populations, while MOMP reactivity peaked at the same level in both AI and TFI populations but did not increase with disease severity. Test populations were standardized by level of reactivity to formalin-fixed Chlamydia trachomatis elementary bodies (EBs) to address whether these associations were reflections of increased overall chlamydial exposure rather than a property specific to Hsp10. Associations between Hsp10 seropositivity and TFI were greater in the EB(+) subgroup while associations among the EB(-) subgroup were diminished. When restricted to the EB(+) subgroups, Hsp60 and MOMP responses in the TFI population did not increase significantly over the level of AI group responses. Thus, among women with similar exposure to chlamydiae, the serologic response to Hsp10 exhibited a stronger correlation with TFI than did the response to Hsp60 or MOMP. These findings support the hypothesis that the serological response to C. trachomatis heat shock proteins is associated with the severity of disease and identifies Hsp10 as an antigen recognized by a significant proportion of women with TFI.

Platelet activation in patients after an acute coronary syndrome: results from the TIMI-12 trial. Thrombolysis in Myocardial Infarction.

UNLABELLED: This study was designed to determine the magnitude and time course of platelet activation during therapy of acute coronary syndromes with an oral platelet antagonist. BACKGROUND: Platelet activation and aggregation are central to the pathogenesis of the acute coronary syndromes (ACS). However, few data are available on levels of platelet activation over time in patients with ACS, especially in the setting of chronic glycoprotein (GP) IIb/IIIa inhibition. METHODS: The Thrombolysis in Myocardial Infarction (TIMI) 12 trial was a phase II, double-blind trial evaluating the effects of sibrafiban, an oral, selective antagonist of the platelet glycoprotein IIb/IIIa receptor in patients stabilized after an ACS. A subset of 90 of the 329 patients in the study had measurement of platelet activation as assessed by the expression of platelet associated P-Selectin on days 0, 7 and 28. Platelet activation was measured in blood samples that were fixed either immediately (spontaneous activation) or after 5 minute incubation with 0, 1 microM or 5 microM ADP in order to assess platelet responsiveness to very low or moderate stimulation. RESULTS: At baseline there was a significant elevation of spontaneous platelet activation as compared to samples obtained from normal donors or from patients who did not have acute coronary syndromes (ACS patients 27.6+/-18.7%, Normal controls 8.5+/-4.4%, Patient controls 10.9+/-7.1%, p < 0.005 for both). In addition, there was a significant decrease in the levels of platelet activation with time during the 28 days of treatment with sibrafiban. Nevertheless, even on day 28, the TIMI-12 patients continued to show elevated platelet activation in comparison to the control groups (p < 0.05 for both). CONCLUSIONS: These results suggest that platelets remain activated long after clinical stabilization post ACS. Although platelet activation decreased after one month of oral GPIIb/IIIa inhibition, levels remained higher than normal, suggesting the need for long-term antiplatelet therapy following ACS.

Antibodies to the chlamydial 60 kilodalton heat shock protein in women with tubal factor infertility.

INTRODUCTION: Fallopian tube damage and subsequent infertility are common sequelae of upper genital tract infection with Chlamydia trachomatis. This fallopian tube damage is thought to be immune mediated. The 60 kilodalton chlamydial heat shock protein (hsp) may be the key antigen associated with this pathogenic response. Our objective was to study the relationship between antibody response to 60 kilodalton chlamydial hsp and tubal factor infertility (TFI). SUBJECTS AND METHODS: Twenty-three women with TFI and 33 women with male factor infertility (controls) were studied. Tubal factor infertility was defined as infertility for one year with hydrosalpinx or distal tubal occlusion. Patients' sera were tested for antibodies to the chlamydial hsp using an enzyme-linked immunosorbent assay (ELISA). A stepwise logistic regression was performed by each patient's age, race/ethnicity, self-reported history of chlamydia infection, gonorrhea, or pelvic inflammatory disease (PID), history of ectopic pregnancy, and antibodies to the chlamydial hsp. RESULTS: Eighteen of the 23 women with TFI had a positive result on the hsp ELISA (78.6%) versus 23.4% of controls. Risk factors for TFI were a history of PID (P = 0.022), "nonwhite" race (P = 0.004), history of ectopic pregnancy (P = 0.027), and antibodies to the 60 kilodalton chlamydial hsp (P < 0.001). CONCLUSIONS: Antibodies to 60 kilodalton chlamydial hsp are strongly associated with TFI.

Peripheral blood manifestations of T(H)2 lymphocyte activation in stable atopic asthma and during exercise-induced bronchospasm.

BACKGROUND: Recently, TH2 lymphocyte activation has been shown to play a key role in initiating and propagating the inflammatory response in asthmatic airways. This is manifest through increased numbers of "activated" CD25-(IL-2R)-bearing T-helper cells and can be seen through the IL-5 driven recruitment of eosinophils and IL-4-mediated B-cell expression of CD23 (low affinity IgE receptor) and ultimately IgE production. OBJECTIVE: To gain a better understanding of the role of immune cells in asthma by describing the peripheral blood immune cell phenotypes in mild atopic asthma. METHODS: We enrolled 13 patients with mild atopic asthma and a group of seven nonatopic, nonasthmatic controls. Objective measures of lung function were obtained. The peripheral blood was analyzed by flow cytometry for specific cellular markers at rest and during the development of exercise induced bronchospasm. RESULTS: At rest the number of CD23-bearing B cells (169/mL versus 117/mL; P = .05) and the number of CD25-bearing T cells (355/mL versus 237/mL; P = .03) were increased in the asthma group. There was a linear relationship between these two lymphocyte subsets and the maximum voluntary ventilation at rest (r = 0.56, P = .01 and r = 0.57, P = .01). With the development of exercise-induced bronchospasm there was a significantly greater increase in CD23-positive B cells (96.7/mL versus 59.7/mL; P = .05) and CD25-positive T cells (111.8/mL versus 45.1; P = .01) in the asthma group. CONCLUSIONS: These data indicate that TH2 lymphocyte activation is manifested by increased numbers of CD23-bearing B cells and CD25-bearing T cells in the peripheral blood of patients with stable mild atopic asthma. Further, these immune cell subsets correlate with markers of resting lung function and increase in the peripheral blood early after the development of exercise-induced bronchospasm.

Genetic control of platelet activation in inbred mouse strains.

Platelet activation in inbred mouse strains was studied using expression of P-selectin as a marker of activated platelets. P-selectin expression in response to no added stimulus (spontaneous activation) or in response to adenosine diphosphate (ADP) and epinephrine or thrombin, was assessed using a flow cytometric assay. Wide variation in the responsiveness of different strains was observed with strains SJL and AKR in particular showing very high levels of spontaneous activation. Genetic studies suggest that this phenomenon is under control of a small number of genes and that the same loci are probably responsible for the high activation of both SJL and AKR. Bone marrow transplant experiments show that the trait is expressed in the platelet itself. Screening of SWXJ and AKXD recombinant inbred lines suggests that one of the responsible genes is located on chromosome 3.

Tumor necrosis factor-alpha response to infection with Chlamydia trachomatis in human fallopian tube organ culture.

OBJECTIVE: Our purpose was to determine whether tumor necrosis factor-alpha is produced in response to infection with Chlamydia trachomatis in the fallopian tube. STUDY DESIGN: Fallopian tubes were harvested at the time of abdominal hysterectomy and processed by standard tissue culture techniques. Tubal segments were inoculated with Chlamydia trachomatis serotype E/UW-5/CX. At 48 hours of incubation supernatant fluid was assayed for tumor necrosis factor-alpha. Tubal segments were stained for chlamydial inclusions and tumor necrosis factor-alpha by use of immunohistochemical techniques. RESULTS: Mean tumor necrosis factor-alpha levels for infected segments were 92.1 +/- 21.3 pg/ml (mean +/- SEM) and for control segments were 61.9 +/- 13.9 pg/ml (p = 0.03 by paired t test). Tumor necrosis factor-alpha was predominantly localized in the tubal epithelium. CONCLUSIONS: Tumor necrosis factor-alpha is produced in response to chlamydial infection by the human fallopian tube. It is an important proinflammatory cytokine and may promote the production of other cytokines and immune-mediated damage of the fallopian tube.

In vivo biotinylation demonstrates that reticulated platelets are the youngest platelets in circulation.

In both mice and humans, a subset of platelets can be identified that shows increased labeling with nucleic acid-specific fluorescent dyes, such as thiazole orange. Termed "reticulated platelets," they have been postulated to be platelets that have recently entered the circulation. Their numbers appear to reflect the rate of new platelet production in a number of clinical and experimental situations. To determine whether reticulated platelets really are the youngest platelets in circulation and to estimate the length of time that they are identifiable after entering the circulation, we have employed a technique of "in vivo biotinylation" in mice that labels the entire cohort of circulating cells with covalently bound biotin. Blood samples can then be double-labeled with fluorescent avidin derivatives and thiazole orange, permitting correlated measurement of both surface biotin content and nucleic acid content. The biotinylation occurs rapidly, is complete within 30 minutes, is stable for several days, and does not appear to alter platelet function. The results show that within 24 hours after in vivo biotinylation, platelets appear in the circulation with decreased levels of biotinylation and that these are the reticulated platelets. The estimated lifespan of reticulated platelets is 1.8 days, and the lifespan of all platelets by this method is 4.5 days, which is in agreement with estimates made by other methods.

Flow cytometric reticulocyte analysis. Multiinstitutional interlaboratory correlation study.

Reticulocyte analysis by flow cytometry offers precision and sensitivity greater than those of conventional morphologic methods and permits derivation of a reticulocyte maturity index. However, interlaboratory variability has not yet been reported. The authors analyzed 310 samples at eight sites using 11 instruments over a 4-month period to examine intermethod and interlaboratory variabilities. Stains included thiazole orange, ethidium bromide, and auramine O. Instruments included models by Coulter, Becton Dickinson, TOA Medical Electronics, and Ortho Diagnostics. The coefficient of variation (CV) among all sites and methods on these samples varied as a function of the reticulocyte percentage, ranging from a mean CV of 69% for samples with < .5% reticulocytes to 24.1% for those with > 2.5% reticulocytes. The best performance was observed with the TOA R-1000 dedicated reticulocyte analyzers, with a mean CV of 18.4% for samples with < .5% reticulocytes and 4.6% for samples with > 2.5% reticulocytes. The reticulocyte maturity index showed comparable intersite precision, with a mean CV of 16% for samples with > 2.5% reticulocytes with multipurpose flow cytometers and a mean CV of 7.3% with the TOA R-1000 instruments. Interclass correlations among all sites ranged from .79 to .99 for the reticulocyte counts and .41 to .88 for the reticulocyte maturity index. The authors conclude that flow cytometric reticulocyte analysis is more precise than manual reticulocyte analysis. With greater automation of this methodology, further interlaboratory standardization of reticulocyte counts and the reticulocyte maturity index can be achieved.