Gamma-carboxylated isoforms of recombinant human protein S with different biologic properties

Human protein S (HPS), a regulator of hemostasis, is a vitamin K- dependent plasma protein with potential clinical utility. We have obtained high-level expression of the cDNA for HPS in two mammalian cell lines. Both cell lines secreted single chain recombinant HPS (rHPS) in serum-free medium as determined by Western blot analysis. The ability of the rHPS from both cell lines to act as a cofactor for human protein C (HPC) was determined; the rHPS secreted from the human 293 cell line had an activity six times that of the rHPS from the AV12-664 Syrian hamster cell line. Furthermore, the relative specific cofactor activity of rHPS from the 293 cell line was actually 2.5-fold higher than that of single-chain human plasma-derived HPS. Essentially all of the rHPS secreted from the 293 cell line exhibited a calcium-dependent elution profile on anion exchange chromatography, whereas only 25% to 35% of the hamster cell-derived rHPS exhibited this profile. However, the calcium-eluted rHPS from the AV12 cell line had a high specific cofactor activity, equivalent to that of the 293-derived rHPS. A NaCl- elutable rHPS fraction (calcium nondependent) was isolated from the recombinant AV12-664 cell line, further purified, and found to have reduced activity, only 40% that of the calcium-dependent rHPS. The only observable difference in the calcium-dependent and nondependent rHPS molecules was in the content of gamma-carboxyglutamic acid (Gla); the calcium-dependent material contained approximately 10 mol Gla/mol protein whereas the calcium-nondependent material contained only approximately 8 mol Gla/mol of protein. In addition, the calcium- nondependent rHPS had reduced ability to interact with phospholipid vesicles as evidenced by an eightfold increase in the apparent kd. Our data demonstrate the isolation of rHPS with high specific activity, and show that a reduction in as few as two Gla residues dramatically decreases its functional cofactor activity for HPC, due to a reduction in ability to interact with the phospholipid bilayer.     

Sildenafil in pediatric pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a life-threatening disease of varied etiologies. Although PAH has no curative treatment, a greater understanding of pathophysiology, technological advances resulting in early diagnosis, and the availability of several newer drugs have improved the outlook for patients with PAH. Sildenafil is one of the therapeutic agents used extensively in the treatment of PAH in children, as an off-label drug. In 2012, the United States Food and Drug Administration (USFDA) issued a warning regarding the of use high-dose sildenafil in children with PAH. This has led to a peculiar situation where there is a paucity of approved therapies for the management of PAH in children and the use of the most extensively used drug being discouraged by the regulator. This article provides a review of the use of sildenafil in the treatment of PAH in children.KEY WORDS: Child, phosphodiesterase (PDE)-5 inhibitor, Pulmonary hypertension therapy     

A totally synthetic lipopeptide-based self-adjuvanting vaccine induces neutralizing antibodies against heat-stable enterotoxin from enterotoxigenic Escherichia coli

ST-based lipopeptide vaccine candidates were constructed in which ST was chemically synthesized and folded into the correct conformation prior to ligation to a module containing a T-helper cell epitope (T(H)) and the Toll-like receptor 2 (TLR2) agonist, S-[2,3-bis(palmitoyloxy)propyl]cysteine (P2C). Two different chemistries, thioether-based and oxime-based, were then used to ligate ST to the lipidated T(H) epitope. The enterotoxic activity of synthetic ST and the ST-based lipopeptide vaccines was determined in mice followed by an evaluation of immunological efficacy. The importance of the fine detail in chemical composition used in vaccine design was demonstrated by the findings that (i) the oxime-based vaccine exhibited little or no toxicity but the thioether-based vaccine, exhibited residual toxicity in suckling mice, (ii) although each of the synthetic vaccines generated specific anti-ST antibodies, it was the low titer antibodies induced by the oxime-based vaccine that demonstrated better neutralizing activity suggesting that the chemical linkage also affects the specificity of antibodies, (iii) the geometric arrangement of ST within a vaccine can profoundly affect the specificity and biological function of the antibodies that are elicited, and (iv) the lipopeptide-based ST vaccine candidate assembled using oxime chemistry induced a better neutralizing antibody response to ST when administered by the mucosal (intranasal) route.     

The impact of implementing national hypertension guidelines on prevalence and workload in primary care: a population-based survey of older people

Recently revised UK and US hypertension guidelines have reduced thresholds for both diagnosis and treatment and differ in their recommendations. We have used data from a random, stratified community-based sample of 4784 people aged 65 years and over to compare the prevalence of treatable hypertension and the potential impact on patients and primary care from using current guidelines. BHS, NICE and JNC7 guidelines were applied to blood pressures obtained from primary care medical records (94%) or measured at a screening clinic (6%). Risk factors were obtained by questionnaire and from medical records, supplemented by epidemiological data. Workload was estimated for a representative practice population of 10 000 patients. Blood pressures were obtained on 4514 patients (94%). Prevalence of treatable hypertension was over 67%. Compared to BHS4, prevalence estimates using NICE guidelines were comparable for men but significantly lower for women (P<0.05). They were significantly higher using JNC7 compared with BHS4 and NICE guidance (P<0.05). A general practice of 10 000 patients could expect 1287 older hypertensive patients using BHS4 guidelines and 1231 patients using NICE guidelines. Under BHS4, an extra 94 patients will require annual, rather than 5-yearly review compared with that using the previous guideline. In conclusion, implementation of BHS4 guidelines, with their revised thresholds for diagnosis, will not add materially to the prevalence of treatable hypertension compared to previous BHS3 guidelines but will have a major impact on practice workload. Use of NICE guidelines in preference to BHS4 will result in GPs treating fewer patients and reviewing untreated patients less often.     

Protection of ovarian function by oral contraceptives in women receiving chemotherapy for Hodgkin's disease

It has been reported by us and by others that after chemotherapy for Hodgkin's disease the ovary contains fewer than 5 primordial and primary follicles per 5 x 5 mm biopsy section. In young women this is associated with premature menopause. We report here that before treatment the tissue contains 18--55 such follicles per biopsy section. When women took combination oral contraceptives throughout the course of MVPP therapy, the posttreatment ovarian biopsy tissue had more than 20 follicles per histologic section. Normal menses were established in the five women who discontinued oral contraceptives at the end of MVPP therapy, and one of them is now pregnant.     

World Workshop on Oral Medicine VI: a systematic review of medication‐induced salivary gland dysfunction

The aim of this paper was to perform a systematic review of the pathogenesis of medication‐induced salivary gland dysfunction (MISGD). Review of the identified papers was based on the standards regarding the methodology for systematic reviews set forth by the World Workshop on Oral Medicine IV and the PRISMA statement. Eligible papers were assessed for both the degree and strength of relevance to the pathogenesis of MISGD as well as on the appropriateness of the study design and sample size. A total of 99 papers were retained for the final analysis. MISGD in human studies was generally reported as xerostomia (the sensation of oral dryness) without measurements of salivary secretion rate. Medications may act on the central nervous system (CNS) and/or at the neuroglandular junction on muscarinic, α‐and β‐adrenergic receptors and certain peptidergic receptors. The types of medications that were most commonly implicated for inducing salivary gland dysfunction were those acting on the nervous, cardiovascular, genitourinary, musculoskeletal, respiratory, and alimentary systems. Although many medications may affect the salivary flow rate and composition, most of the studies considered only xerostomia. Thus, further human studies are necessary to improve our understanding of the association between MISGD and the underlying pathophysiology.     

Respiratory infection and otitis media visits in relation to pneumococcal conjugate vaccine use in Saskatchewan

BACKGROUND:

In Saskatchewan, pneumococcal conjugate vaccination (PCV) was offered to high-risk children in 2002 and to all infants in 2005.

OBJECTIVE:

To describe trends in the frequency of medical visits for lower respiratory tract infection (LRI) and otitis media (OM) in relation to PCV use during the period 1990 to 2008.

METHODS:

Statistics regarding the number of children covered by the health insurance plan, PCV administration, and medical visits with a diagnostic code associated with LRI and OM were provided by Saskatchewan Health. Monthly rates were analyzed using dynamic state space models.

RESULTS:

In all series, there was a marked seasonal cycle and some higher-than-expected winter peak values, possibly associated with epidemics of specific respiratory viruses. Three abrupt decreases in baseline rate were observed for LRI and the final one, in February 2007, could be related to the increased proportion of children vaccinated with PCV. There was no statistical correlation between PCV use and OM visit frequency.

CONCLUSION:

Many environmental, biological and administrative factors may influence health services use, and an effect of low magnitude of a particular vaccine pertaining to nonspecific outcomes could be obscured in time-series analyses.     

Early sexual initiation: the role of peer norms

OBJECTIVE: To elucidate which components of peer norms influence the process of sexual initiation for young adolescents. Design. Prospective cohort study. Setting. Fourteen elementary and middle schools in an urban public school district. Participants. The 1389 sixth-grade students who completed the questionnaire at the beginning (time 1) and at the end (time 2) of the school year comprise the study sample. Mean age at time 1 was 11.7 years. RESULTS: Of students entering the sixth grade, 30% (n = 416) reported having already initiated sexual intercourse, 5% (n = 74) reported initiating sexual intercourse during the sixth-grade school year (initiated group), and 63% (n = 873) reported not having initiated sexual intercourse by the end of the sixth-grade school year (never group). Demographic comparisons revealed that students in the initiated group were significantly more likely than students in the never group to be older (11.9 years vs 11.6 years), male (58% vs 37%), African-American (70% vs 51%), attending a poorer school (87% vs 85%), and living in an area with a high proportion of single-parent families (45% vs 41%). Self-reports and reports of peers' participation in nonsexual risk behaviors were more common for students in the initiated group. Students in the initiated group were more likely than students in the never group to perceive: 1) a high prevalence of sexual initiation among peers; 2) social gains associated with early sexual intercourse; and 3) younger age of peers' sexual initiation. Students in the never group were more likely to believe that sexually-experienced 12-year-old boys would be negatively stigmatized compared with students in the initiated group. Three predictive models were developed to test the relationship between peer norms and the process of initiation. These models demonstrate that the strongest predictor of sexual initiation in sixth grade is having high intention to do so at the beginning of sixth grade. The strongest predictor of high intention is belief that most friends have already had sexual intercourse. Perceptions of social gain and stigma for sexually-experienced 12-year-old boys act independently of intention to decrease risk of early sexual initiation. CONCLUSION: Early sexual intercourse is not an unplanned experience for many teens. Decisions about initiation are strongly bound to social context with peers playing an important role in creating a sense of normative behavior. Specific components of peer norms impact the process of sexual initiation in both positive and negative ways. Interventions aimed at delaying the onset of sexual initiation need to focus on cohort norms as well as on an individual's perceptions and behaviors.