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.     

Echinococcal disease and mycobacterial infection.

The clinical and radiological features of five patients with echinococcal disease in whom mycobacterial infection was also present are described. Three had clinically significant disease due to Mycobacterium tuberculosis (two patients) and M. fortuitum-chelonae (one patient), whilst in two others secondary infection with M. gordonae had occurred. In all cases the mycobacterium infection was found within or close to the anatomical confines of the echinococcal disease, as determined clinically, radiologically or histologically, which suggested a saprophytic-supportive role of the hydatid cyst for the mycobacterium to survive. Mycobacterial disease should be borne in mind in patients with echinococcal disease who remain unwell despite apparently effective treatment for the hydatid cyst.     

Acute dehydrating disease caused by Vibrio cholerae serogroups O1 and O139 induce increases in innate cells and inflammatory mediators at the mucosal surface of the gut

BACKGROUND AND AIMS: The general concept is that as Vibrio cholerae is not invasive, it mediates a non-inflammatory type of infection. This is being re-evaluated based on available data that natural cholera infection or cholera toxin induces a Th2-type of immune profile and stimulates the humoral immune response, innate cells, and mediators in the host. METHODS: To perform a comprehensive analyses of the inflammatory components, we studied mucosal biopsies from patients, both adults and children with acute watery diarrhoea caused by V cholerae O1 and O139. Patients with cholera, adults (n = 30) and children (n = 18), as well as healthy controls (n = 24) were studied. Histochemical, immunohistochemical, and ultrastructural studies were carried out to elucidate the contribution of the different factors using paraffin and frozen duodenal and/or rectal sections as appropriate. Samples were collected during the acute stage and during early and/or late convalescence. RESULTS: Following natural cholera infection, patients responded with increases in neutrophil polymorphs during the acute stage (p<0.001) compared with healthy controls whereas mucosal mast cells (MMC) (p = 0.008) and eosinophils (p = 0.034) increased in the gut during convalescence. Electron microscopic analyses of duodenal biopsies from adult patients showed increased piecemeal degranulation in both MMC and eosinophils and accumulation of lipid bodies in MMC. Duodenal biopsies from V cholerae O1 infected patients showed upregulation of myeloperoxidase, lactoferrin, PGHS-1, SCF, tryptase, tumour necrosis factor alpha, alpha-defensin, and eotaxin during the acute stage and chymase, interleukin 3 and major basic protein during convalescence. CONCLUSION: We have shown that innate cells and their mediators are upregulated in acute watery diarrhoea. These cells and factors of the innate arm may be important in the host's defence against cholera. Such effects may need to be simulated in a vaccine to achieve long lasting protection from cholera.     

Trophoblasts reduce the vascular smooth muscle cell proatherogenic response

Maternal spiral artery remodeling is the consequence of controlled trophoblast invasive interaction with the maternal cellular environment and is fundamentally important for successful placentation. In preeclampsia, trophoblast invasion is shallow, remodeling is incomplete, and vessels develop an inflammatory appearance, termed "acute atherosis." We noted that, in our preeclampsia, human renin-human angiotensinogen transgenic rat model, complement component 3 (C3), and tumor necrosis factor-alpha were upregulated and heavily expressed in atherotic uteroplacental vessels. We next used coculture involving human trophoblasts, rat vascular smooth muscle cells (VSMCs), and human VSMCs to observe VSMC-trophoblast regulatory interactions. Tumor necrosis factor-alpha induced complement C3 and interleukin-6 expression in VSMCs. We found that trophoblasts were able to reduce VSMC C3 and interleukin-6 expression after the VSMCs were stimulated with tumor necrosis factor-alpha. However, a direct VSMC-trophoblast cell-cell contact was necessary for this anti-inflammatory response. We also studied double-transgenic VSMCs that express inflammatory components and exhibit accelerated proliferation ("synthetic" phenotype). Trophoblasts could not downregulate C3 in these cells. We then examined uteroplacental tissues from preeclamptic and control patients. In control deciduas, only traces of C3 staining were observed, and vessels were thin walled without thrombus formation. In preeclampsia, the decidual vessels showed atherosis, thrombus formation, and C3 expression. Our data suggest that fetally derived trophoblasts require direct cell-cell contact with maternally derived VSMCs to downregulate VSMC C3 and interleukin-6 expression and to avoid atherosis. The findings also implicate C3 in the placental vasculopathy observed in preeclampsia.     

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.     

Differential expression of AT1 receptors in the pituitary and adrenal gland of SHR and WKY

The renin-angiotensin (ANG) system has been implicated in the development of hypertension in spontaneously hypertensive rats (SHR). Because SHR are more susceptible to stress than normotensive Wistar-Kyoto rats (WKY), we measured the mRNA expression of AT1A, AT1B, and AT2 receptors in the hypothalamo-pituitary-adrenal (stress) axis of male SHR in comparison to age-matched WKY at prehypertensive (3 to 4 weeks), developing (7 to 8 weeks), and established (12 to 13 weeks) stages of hypertension. AT1A receptor mRNA was mainly expressed in the hypothalamus and adrenal gland. AT1B receptor mRNA was detected in the pituitary and adrenal gland. AT2 receptor mRNA was prominent only in the adrenal gland. When compared with WKY, SHR showed increased AT1A receptor mRNA levels in the pituitary gland at all ages in contrast to reduced pituitary AT1B receptor mRNA levels. In the adrenal gland of SHR, AT1B receptor mRNA levels were decreased at the hypertensive stages when compared with WKY. The reduced expression of adrenal AT1B receptor mRNA was localized selectively in the zona glomerulosa by in situ hybridization. No differences were observed between WKY and SHR in the expression of hypothalamic ANG receptors. ANG significantly increased plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone in dexamethasone-treated SHR but not in WKY. The aldosterone response to ANG was similar in SHR and WKY. Our results suggest a differential gene expression of AT1A and AT1B receptors in the hypothalamo-pituitary-adrenal axis of SHR and normotensive WKY and imply the participation of AT1 receptors in an exaggerated endocrine stress response of SHR to ANG.     

Space closing versus space opening for bilateral missing upper laterals – aesthetic judgments of laypeople: a web-based survey

Objective: To investigate the opinions of laypeople regarding the aesthetic outcome of treating patients with developmental absence of both maxillary lateral incisors using either orthodontic space closure (OSC) or space opening and prosthetic replacement (PR). Design: Cross sectional, web-based survey. Methods: A panel of five orthodontists and five restorative dentists examined post-treatment intra-oral images of 21 patients with developmental absence of both upper lateral incisors. A consensus view was obtained about the 10 most attractive images (5 OSC; 5 PR). The 10 selected images were used in a web-based survey involving staff and students at the University of Sheffield. In the first section, the participants were asked to evaluate the attractiveness of the 10 randomly arranged single images using a 5-point Likert scale. In the second section, an image of OSC was paired with an image of PR according to their attractiveness ranking by the clinician panel, and the participants were asked to indicate which of the two images they preferred. Results: The survey received 959 completed responses with 9590 judgements. The images of OSC were perceived to be more attractive (mean rating 3·34 out of 5; SD 0·56) compared with the images of PR (mean rating 3·14 out of 5; SD 0·58) (mean diff 0·21; P?Conclusion: Space closing was perceived to be more attractive than space opening by lay people. The findings have implications for advising patients about the best aesthetic outcome when both maxillary lateral incisors are missing.     

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.     

Cytomegalovirus detection by biotinylated DNA probes

Clinical specimens from 317 patients suspected of cytomeglovirus infection were examined by immunofluorescence (IF) using monoclonal antibodies and by a biotinylated DNA probe kit after cell culture isolation. Of the 317 samples, 68 were positive by culture isolation. Of these 67 were IF positive when the cytopathic effect (CPE) was 1⁺ or less, whereas 56 gave positive results with DNA probes when the CPE was 2⁺. A further 83 specimens were examined directly by immunoperoxidase histopathology (IHP), IF and the DNA probe kit: 26 of these were positive by IHP examination, 25 by IF and only 6 by DNA probes. The sensitivity of the DNA probe kit was not satisfactory when the clinical tissue specimens were directly examined. However, the sensitivity improved considerably to 82% if the specimens were propagated first in cell culture. The IF method detected the virus before and after cell culture isolation equally well (96%–98.5%). Compared to the IF method, the DNA probe kit is costly and requires more labor and time.This paper was presented in part at the 88th annual meeting of the American Society for Microbiology, Miami Beach, FL, USA, 1988     

Oral cholera vaccination strategy: Self-administration of the second dose in urban Dhaka,Bangladesh

Cholera remains a major public health problem in many developing countries including Bangladesh. The oral cholera vaccine (OCV) is now considered a key component of the public health response to cholera. Although maintaining cold chain and organizing human resource are the major challenges of vaccine delivery to the community. Here we applied an innovative approach to second dose OCV delivery to minimize financial and logistic burdens. The purpose of this study was to assess the feasibility and compliance of second dose self-administration when the second dose was provided in a plastic bag to first dose vaccine recipients as OCV is stable for up to 42?days at ambient temperatures. We aimed to deploy vaccines (N?=?112,000) left over from other studies to 56,000 people aged?≥?one year living in Mirpur, Dhaka to see the feasibility of self-administration strategy. During vaccination, the first OCV dose (OCV1) was given from fixed sites and the second dose (OCV2) was provided in a plastic zip-lock bag for the participant to take the vaccine two weeks later at home. Participants were instructed to keep the vaccine away from light and in a dry cool place. Empty vials were collected following the end date of the scheduled second vaccination. Of the targeted population, 41,694 (74%) received the first OCV dose whereas an estimated 38,852 (93% of those receiving the first dose) received the second dose which represents a 7% drop out rate from OCV1 to OCV2. However the average two dose coverage was 69%. A survey of a subsample 2990 (from 8551) randomly selected households revealed that almost all respondents (98.75%) appreciated this new self-administration strategy and considered the strategy to be more practical and convenient than the usual method. This simplified, self-administered delivery strategy provides an ideal alternative for second-dose OCV delivery in hard-to-reach populations and resource-poor settings.