Interobserver variation in the reporting of cervical colposcopic biopsy specimens: comparison of grading systems.

AIMS: To assess interobserver variation in reporting cervical colposcopic biopsy specimens and to determine whether a modified Bethesda grading system results in better interobserver agreement than the traditional cervical intraepithelial neoplasia (CIN) grading system. METHODS: One hundred and twenty five consecutive cervical colposcopic biopsy specimens were assessed independently by six histopathologists. Specimens were classified using the traditional CIN grading system as normal, koilocytosis, CIN I, CIN II, or CIN III. The specimens were also classified using a modified Bethesda grading system as either normal, low grade squamous intraepithelial lesion (LSIL) or high grade squamous intraepithelial lesion (HSIL). Participants were also asked to categorise biopsy specimens by the CIN system with the addition of the recently proposed category "basal abnormalities of uncertain significance (BAUS)". The degree of agreement between participants was assessed by kappa statistics. RESULTS: Using the CIN system, interobserver agreement was generally poor: unweighted and weighted kappa values between individual pairs of observers ranging from 0.05 to 0.34 (average 0.20) and from 0.20 to 0.54 (average 0.36), respectively. With the modified Bethesda system, interobserver agreement was better but still poor: unweighted and weighted kappa values ranging from 0.15 to 0.58 (average 0.30) and from 0.21 to 0.61 (average 0.36), respectively. There was little or no agreement between observers in the diagnosis of BAUS. CONCLUSIONS: Interobserver agreement in the reporting of cervical colposcopic biopsy specimens using the CIN grading system is poor. Agreement, while still poor, is better when a modified Bethesda grading system is used. There is little or no consensus in the diagnosis of BAUS.     

Randomized controlled trial of brief cognitive behaviour therapy versus treatment as usual in recurrent deliberate self-harm: the POPMACT study

BACKGROUND: We carried out a large randomized trial of a brief form of cognitive therapy, manual-assisted cognitive behaviour therapy (MACT) versus treatment as usual (TAU) for deliberate self-harm. METHOD: Patients presenting with recurrent deliberate self-harm in five centres were randomized to either MACT or (TAU) and followed up over 1 year. MACT patients received a booklet based on cognitive behaviour therapy (CBT) principles and were offered up to five plus two booster sessions of CBT from a therapist in the first 3 months of the study. Ratings of parasuicide risk, anxiety, depression, social functioning and global function, positive and negative thinking, and quality of life were measured at baseline and after 6 and 12 months. RESULTS: Four hundred and eighty patients were randomized. Sixty per cent of the MACT group had both the booklet and CBT sessions. There were seven suicides, five in the TAU group. The main outcome measure, the proportion of those repeating deliberate self-harm in the 12 months of the study, showed no significant difference between those treated with MACT (39%) and treatment as usual (46%) (OR 0.78, 95% CI 0.53 to 1.14, P=0.20). CONCLUSION: Brief cognitive behaviour therapy is of limited efficacy in reducing self-harm repetition, but the findings taken in conjunctin with the economic evaluation (Byford et al. 2003) indicate superiority of MACT over TAU in terms of cost and effectiveness combined.     

Immunohistochemical evidence for mesothelial origin of paratesticular adenomatoid tumour

AIMS: To investigate the histogenesis of paratesticular adenomatoid tumour by use of immunohistochemical markers for a variety of carcinomas and mesothelioma. METHODS AND RESULTS: Immunohistochemical staining of sections from 12 cases of paratesticular adenomatoid tumour was undertaken using primary antibodies to antigens expressed by benign epithelial cells and carcinoma (cytokeratin AE1/AE3, cytokeratin 34ssE12, epithelial membrane antigen, MOC-31, Ber-EP4, CEA, B72.3, LEA.135, Leu M1), stromal and vascular markers (vimentin, CD34, factor VIII), and mesothelioma-associated antigens (thrombomodulin, HBME-1, OC 125) and p53 protein. There was absence of immunohistochemical expression of epithelial/carcinoma markers MOC-31, Ber-EP4, CEA, B72.3, LEA.135, Leu M1 and to factor VIII and CD34. All tumours expressed cytokeratin AE1/AE3, epithelial membrane antigen and vimentin, with weak expression of cytokeratin 34ssE12 in 25% of tumours. Each tumour showed expression of thrombomodulin, HBME-1 and OC 125 in a membranous distribution. p53 protein expression was not detected. CONCLUSIONS: The immunohistochemical profile of paratesticular adenomatoid tumour is strongly supportive of a mesothelial cell origin.     

Flash labelling of S-phase cells in short-term organ culture of normal and pathological human endometrium using bromodeoxyuridine and tritiated thymidine

Normal hyperplastic and malignant endometrial specimens were labelled in vitro with bromodeoxyuridine (BrdU). S-phase cells were stained after DNA denaturation, using a monoclonal antibody to BrdU and an indirect immunoperoxidase method. Various conditions for BrdU uptake, DNA denaturation, and staining were tested. BrdU labelling was compared with autoradiography using tritiated thymidine, with good correlation. Glandular labelling indices of proliferative endometrium were significantly higher than both secretory and hyperplastic endometrium but were similar to carcinoma. Stromal labelling showed the same trend but the differences were not statistically significant.     

Mobilization of calcium by the brief application of oxytocin and prostaglandin E2 in single cultured human myometrial cells.

Intracellular calcium ([Ca2+]i) mobilization was studied in single cultured human myometrial cells in response to the agonists oxytocin and prostaglandin E2 (PGE2) using the fluorescent dye Fura-2. Oxytocin and PGE2 applications were associated with an increase in [Ca2+]i, although there was a marked intercell variation in the amplitude of the agonist-induced response. Removal of extracellular calcium ([Ca2+]o) reduced the oxytocin-induced rise and abolished the PGE2-induced rise in [Ca2+]i, thereby demonstrating that oxytocin but not PGE2 can mobilize intracellular stores of calcium. In nominally calcium-free medium, [Ca2+]i was not increased by PGE2 but subsequent application of oxytocin increased [Ca2+]i, thereby demonstrating that, within a single cell, calcium stores were mobilized by oxytocin and not PGE2. The intracellular calcium stores were completely depleted by a single application of oxytocin and not replenished in the absence of [Ca2+]o. Perfusion with calcium-containing medium for 100 s enabled store refilling. Cell depolarization by 140 mM-K+ caused a transient increase followed by a sustained elevation of [Ca2+]i on which were superimposed small fluctuations. Oxytocin caused an influx of calcium in cells depolarized by K+. This was more marked than that obtained with PGE2.     

E. coli-expressed recombinant norovirus capsid proteins maintain authentic antigenicity and receptor binding capability

The baculovirus expression system has been widely used to produce the capsid proteins of Norovirus (NV) and the proteins form virus-like particles (VLPs) that are useful in many studies, such as immunology, diagnosis, and host-receptor interaction. We report here the application of the E. coli expression system in the production of recombinant NV capsid proteins. In a direct comparison of a previous well-characterized NV strain (VA387), we have demonstrated that the E. coli-expressed capsid proteins maintain the same antigenicity and receptor binding specificity as that of the baculovirus-expressed capsid, although the E. coli-expressed VA387 proteins did not form VLPs. Using the E. coli-expression system, we characterized the receptor-binding patterns of three additional NV strains (OIF1998, Parris Island and VA115), in which OIF1998 binds to HBGA of nonsecretors but did not bind or binds weakly to the HBGA of secretors, as seen in strain VA207. Parris Island binds to HBGA of types A and B but not type O secretors and nonsecretors. VA115 did not show specific binding to any A, B, O secretor nor nonsecretor, which is also observed when the capsid protein of this strain was expressed in baculovirus. Our data indicate that VLP formation is not required for receptor binding, and that the bacteria expression system offers a simple alternative for large production of NV capsid protein for various research purposes, particularly for strains generating low yields in the insect cells.     

Relationship between ROS production, apoptosis and DNA denaturation in spermatozoa from patients examined for infertility

BACKGROUND: The aim of this study was to examine the role of apoptosis and reactive oxygen species (ROS) in inducing DNA damage in ejaculated spermatozoa. METHODS: We examined ejaculated spermatozoa from 31 patients examined for infertility and 19 healthy donors for apoptosis, production of ROS and DNA damage using annexin V binding, chemiluminescence assay and sperm chromatin structure assay. RESULTS: The percentage of spermatozoa that underwent apoptosis in the whole ejaculate and mature fraction was higher in the patients than in the donors (P<0.001 and P=0.009, respectively). Levels of ROS in the whole ejaculate and immature fraction were higher in the patients than in the donors (P=0.002 and P=0.009). Apoptosis was significantly correlated with ROS within patients in the whole ejaculate [r (95% confidence interval)=0.53 (0.19-0.86)] and in the mature [0.71 (0.39-1.00)] and immature spermatozoa [0.75 (0.45-1.00)]. Only apoptosis and the DNA fragmentation index (DFI) were significantly correlated within patients in the whole ejaculate [0.57 (0.18-0.97)]. CONCLUSIONS: DNA damage may be induced by oxidative assault. Apoptosis may not contribute significantly to the DNA damage.     

Phase I testing of a malaria vaccine composed of hepatitis B virus core particles expressing Plasmodium falciparum circumsporozoite epitopes

We report the first phase I trial to assess the safety and immunogenicity of a malaria vaccine candidate, ICC-1132 (Malarivax), composed of a modified hepatitis B virus core protein (HBc) containing minimal epitopes of the Plasmodium falciparum circumsporozoite (CS) protein. When expressed in Escherichia coli, the recombinant ICC-1132 protein forms virus-like particles that were found to be highly immunogenic in preclinical studies of mice and monkeys. Twenty healthy adult volunteers received a 20- or a 50-microg dose of alum-adsorbed ICC-1132 administered intramuscularly at 0, 2, and 6 months. The majority of volunteers in the group receiving the 50-microg dose developed antibodies to CS repeats as well as to HBc. Malaria-specific T cells that secreted gamma interferon were also detected after a single immunization with ICC-1132-alum. These studies support ICC-1132 as a promising malaria vaccine candidate for further clinical testing using more-potent adjuvant formulations and confirm the potential of modified HBc virus-like particles as a delivery platform for vaccines against other human pathogens.     

Effect of pituitary gonadotrophins on proliferation of primary cultures of human ovarian stroma

Proliferation of ovarian stromal cells is a common phenomenon in peri- and post-menopausal ovaries. It is generally assumed to be secondary to the rise in circulating gonadotrophins at the menopause, though the process by which it occurs is poorly understood. This study aimed to examine the effect of menopausal levels of pituitary gonadotrophins on the growth of primary cultures of ovarian stroma. A culture system was developed using primary explants of ovarian stroma on a collagen substrate. The effect of follicle stimulating hormone (FSH; 10(-5) g/l) and luteinizing hormone (LH; 10(-5) g/l) on the proliferation of cultures derived from the cortices and medullae of ten ovaries was evaluated using a dual radiothymidine labelling technique. FSH was stimulatory to cortical cultures from 9/10 ovaries and medullary cultures from 7/10 ovaries, while LH was stimulatory to cortical cultures from 6/9 ovaries and medullary cultures from 5/10 ovaries. The responsiveness of the cultures did not correlate with the degree of hyperplasia in vivo. This study demonstrates that pituitary gonadotrophins may modulate the growth of stromal cells in culture, and thus may play a role in the process whereby stromal proliferation occurs in peri- and post-menopausal ovaries.