Development of a standardized low-dose double-blind placebo-controlled challenge vehicle for the EuroPrevall project.

BACKGROUND: Double-blind placebo-controlled food challenge (DBPCFC) is the gold standard for diagnosing food allergy. Standardized materials and protocols are essential for comparing DBPCFC results for multicentre studies such as EuroPrevall. This required the development and piloting of a standardized vehicle and low-dose protocol for confirming food allergy and determination of minimum eliciting doses (MEDs). METHODS: A low-dose DBPCFC protocol was developed, with eight titrated protein doses from 3 µg to 1 g. This was delivered using a simple, microbiologically stable food base incorporating allergenic food ingredients manufactured at three sites and centrally distributed to clinical centres. Allergen blinding was assessed by a professional sensory testing panel using a triangle test. Homogeneity and allergen content were confirmed by ELISA and clinical efficacy was assessed in a pilot study, using celeriac and hazelnut as exemplars. RESULTS: Celeriac and hazelnut ingredients were sufficiently blinded in the dessert. The dessert meals were successfully piloted with hazelnut in allergy clinics in Spain, the Netherlands and Italy and with celeriac and hazelnut in Zurich. The challenges elicited a range of subjective and objective reactions ranging in severity from mild itching of the oral mucosa to bronchospasm. CONCLUSIONS: A standardized challenge vehicle proven to sufficiently blind processed, powdered hazelnut and celeriac ingredients and that can be reproducibly manufactured has been developed. This pilot study shows that the vehicle is promising for the confirmation of food allergy and determination of MEDs in adults and children with body weight >28.8 kg (approximately 7-11 years old).

Value of mental health first aid training of advisory and extension agents in supporting farmers in rural Queensland.

INTRODUCTION: This study was a pilot project responding to the increasing levels of stress, depression and other mental health issues in Australian rural areas resulting from prolonged drought and a changing economic and social environment. METHODS: Thirty-two Advisory and Extension Agents (AEAs) attended a training course held in 2007 and 2008 in Queensland, Australia. A year after the training, data was collected to determine its value. Interviews were conducted with course participants and their supervisors and focus groups were held with stakeholders (farmers, agency staff and health professionals). RESULTS: The findings show that Mental Health First Aid training improved the participants' confidence level and their knowledge of mental health issues and increased their empathy toward persons with mental health problems. Furthermore, providing training on mental health issues to AEAs was perceived by stakeholders to be beneficial to both farmers and AEAs. CONCLUSION: This study demonstrated that stakeholders and course participants see this type of training as very much needed and highly beneficial. Further, providing training in mental health issues to rural service providers can be very beneficial to their farmer clients and their social network.

Do not be alarmed, the patient is monitored.

Many patients are believed to be at risk of dysrhythmias and are felt to require cardiac monitoring. These patients may not be deemed ill enough to occupy a high dependency or critical care bed and are monitored on general wards. Monitoring policies vary widely not only between institutions, but also between individual medical staff. These variations occur due to differing availability of resources and due to the lack of consensus regarding the risk for an individual patient. There is no clear evidence that monitoring patients outside high dependency areas is of benefit; inappropriate use of monitoring may actually increase patient risk.

Paediatricians' perception of the use of extracorporeal membrane oxygenation to treat meconium aspiration syndrome.

This questionnaire study was designed to determine how consultant paediatricians perceived the value of extracorporeal membrane oxygenation (ECMO) in the treatment of meconium aspiration syndrome (MAS). A significant number believed that the survival of infants with MAS who received ECMO was lower than reported. Early discussion with an ECMO centre will ensure appropriate timing of referral.

Images in cardiology: left ventricular free wall rupture.

Left ventricular free wall rupture is thought to account for approximately 15% of deaths following acute myocardial infarction (MI). We present a case of left ventricular free wall rupture in a 64-year-old man following a lateral MI. The diagnosis was made with 2D and colour flow Doppler echocardiography. Unfortunately the patient died before emergency surgery could be performed.

A preterm baby with Omenn syndrome.

UNLABELLED: A preterm baby born with scaly skin who later developed recurrent infections and was subsequently diagnosed to have Omenn syndrome is presented. CONCLUSION: Any baby with ichthyotic skin and recurrent infections should have immunodeficiency considered in the differential diagnosis.

Coordinated modulation of the fibroblast growth factor dual receptor mechanism during transformation from human colon adenoma to carcinoma.

Basic fibroblast growth factor (bFGF) is dependent on heparan sulphate for its ability to activate the cell surface signal transducing receptor. We have investigated the FGF dual receptor mechanism in a novel model of the transformation from human colon adenoma to carcinoma in vitro. Reverse transcription-polymerase chain reaction showed that mRNA for FGF receptors 1 and 2 were expressed in both the adenoma and carcinoma cells whereas immunocytochemistry showed that the expression of the FGF R1 was reduced significantly in the carcinoma cells. We have reported previously that the composition and sequence of human colon adenoma and carcinoma heparan sulphate (HS) differ in a defined and specific manner. The functional significance of these changes was assessed by affinity co-electrophoresis, which showed that the affinity of adenoma HS for bFGF was 10-fold greater than that of the carcinoma HS (Kd 220 nM vs. 2493 nM, respectively). In addition, Northern studies of the expression of syndecan 1 and 4 mRNA showed that proteoglycan core protein expression was reduced significantly in the carcinoma cells. These findings were associated with a reduced biological response to bFGF in the carcinoma cells that could be partially reversed by the addition of exogenous heparin, suggesting that both the proteoglycan and signal transducing receptor control the cells' response to bFGF.

Effect of Bifidobacterium longum and inulin on gut bacterial metabolism and carcinogen-induced aberrant crypt foci in rats.

The effect of Bifidobacterium longum (4 x 10(8) viable cells/g diet) and a derivative of inulin ('Raftiline HP'; 5% w/w in diet) on colonic aberrant crypt foci (ACF) induced by the colon carcinogen azoxymethane (AOM) has been studied. The concentration of ammonia, a putative tumour promoter produced by bacterial degradation of protein and urea, and the activities of certain bacterial enzymes thought to play a role in colon carcinogenesis, beta-glucuronidase and beta-glucosidase were also assayed. Consumption of either B. longum or inulin was associated with a decrease (26 and 41%, respectively) in AOM-induced small ACF (i.e. those comprising 1-3 aberrant crypts per focus). Combined administration of the bifidobacterium and inulin resulted in more potent inhibition of ACF than administration of the two separately, achieving 80% inhibition of small ACF. Furthermore, the combined administration significantly decreased the incidence (by 59%) of large ACF (>4 aberrant crypts per focus), which are considered to be predictive of eventual tumour incidence. Since the dietary treatments were started 1 week after the carcinogen dose, the results suggest that B. longum and inulin may be affecting the early promotion phase of the carcinogenic process. Consumption of diets containing B. longum, inulin or both were also associated with decreases in beta-glucuronidase activity and ammonia concentration in the caecal contents. Both these factors have been associated with carcinogenesis of the colon in experimental animal models. In rats given inulin-containing diets (with or without B. longum) an increase in caecal wt and beta-glucosidase activity and a decrease in caecal pH were observed. The results suggest that consumption of B. longum or inulin was associated with potentially beneficial changes in caecal physiology and bacterial metabolic activity in relation to tumour risk and in the incidence of putative preneoplastic lesions in the colon. The results also indicated that combined treatment with the two agents was more effective in reducing colonic lesions.

The predictive value of the sperm-cervical mucus interaction test on the outcome of in vitro fertilization and ovulation induction combined with intrauterine insemination.

We investigated the effect of the sperm-cervical mucus penetration tests (SPT) on the fertilization rate (FR) and pregnancy rate (PR) in patients treated with either in vitro fertilization (IVF) or ovulation induction combined with intrauterine insemination (OI + IUI). Infertile couples where the women had normal ovarian function and a normal pelvis at laparoscopy and her partner had normal seminology who had failed at least 2 SPTs were treated with either IVF or OI + IUI. These patients were compared with similar couples in whom SPTs were satisfactory (SPT/ve). Group A (SPT+ve) consisted of 46 patients who underwent 78 treatment cycles of IVF and Group B (SPT-ve) comprised 31 patients who underwent 35 IVF cycles. Group C (SPT/ve) consisted of 39 patients who underwent 84 treatment cycles with OI + IUI, and Group D (SPT-ve) consisted of 15 patients who underwent 37 cycles with the same treatment. In patients treated with IVF, the FR and PR per embryo transfer were 77.0% and 20.0% respectively in Group A, and 64.0% and 22.6% respectively in Group B. The difference in FRs was statistically significant (p > 0.001) but there was no difference in the PRs. In patients treated with OI + IUI, the PR per cycle were 22.0% in Group C and 16.2% in Group D. These results indicate that SPT failure was associated with a lower FR in IVF but this did not affect the PRs. Similarly there was no difference in PRs following OI + IUI.

Identification of genetic changes associated with drug resistance by reverse in situ hybridization.

The molecular cytogenetic techniques of comparative genomic hybridization (CGH) and reverse in situ hybridization (REVISH) allow the entire genomes of tumours to be screened for genetic changes without the requirement for specific probes or markers. In order to define the ability of REVISH to detect and map regions of amplification associated with drug resistance, we investigated a panel of cell lines selected for resistance to doxorubicin and intrinsic sensitivity to topoisomerase II-inhibitory drugs. We have defined a modified REVISH protocol, which involves double hybridizations with genomic DNA from the test cell lines and chromosome-specific whole chromosome paints to identify the chromosomes to which the amplicons localize. Sites of amplification are then mapped by fractional length measurements (Flpter), using published genome databases. Our findings show that amplification of the topoisomerase II alpha gene is readily detected and mapped, as is amplification of the MDR and MRP loci. Interestingly, REVISH detected a new amplicon in the doxorubicin-resistant lung cancer cell line, GLC4-ADR, which mapped to chromosome 1q. REVISH is therefore ideally suited to characterize genetic changes specific for drug resistance within a background of genetic anomalies associated with tumour progression.

Purified urinary follicle stimulating hormone induces different hormone profiles compared with menotrophins, dependent upon the route of administration and endogenous luteinizing hormone activity.

The effects of treatment of patients with gonadotrophin-releasing hormone analogue (GnRHa) combined with purified follicle stimulating hormone (FSH) for in-vitro fertilization (IVF) were investigated in detail to determine the influences of different administration routes and the degree of suppression of luteinizing hormone (LH). Responses to exogenous gonadotrophins were studied in infertile women (n = 60) with normal menstrual rhythm whose endogenous gonadotrophin activity was suppressed using a GnRHa in a long protocol. They were randomized to receive i.m. administration of human menopausal gonadotrophins (HMGim, Pergonal) or purified follicle stimulating hormone (FSH, Metrodin High Purity) administered either i.m. (MHPim) or s.c. (MHPsc). Responses were assessed by measuring plasma FSH, LH, oestradiol, testosterone and progesterone. After stimulation day 4, the MHPsc group showed significantly higher circulating concentrations of FSH than either the MHPim or HMGim group. However, the HMG group showed significantly higher oestradiol concentrations after stimulation day 5 than either MHP group. The differences in circulating oestradiol concentrations in the MHP-treated patients appeared to be strongly influenced by the mean circulating concentrations of LH in the follicular phase. The patients who showed mean follicular phase LH concentrations of < 1 IU/l showed longer follicular phases, lower circulating oestradiol and testosterone concentrations and also lower follicular fluid concentrations of oestradiol and testosterone, indicating a reduction in the normal follicular metabolism of progesterone to androgens and oestrogens under these conditions. This group of patients also showed longer follicular phases, which may have consequences for future clinical management.

Selection of a subpopulation with fewer DNA topoisomerase II alpha gene copies in a doxorubicin-resistant cell line panel.

A panel of doxorubicin-resistant sublines of the human small-cell lung carcinoma cell line GLC4 displays decreasing DNA topoisomerase II alpha (TopoII alpha) mRNA levels with increasing resistance. In the present study we describe how this decrease may be regulated. No significant differences in TopoII alpha mRNA stability or gene arrangement were found, using mRNA slot-blotting and Southern blotting, in the most resistant cell line compared with the parental cell line. To investigate if TopoII alpha gene copy loss contributed to the mRNA decrease, fluorescence in situ hybridisation using a TopoII alpha-specific probe was performed. During doxorubicin resistance development, the composition of the population in each cell line shifted with increasing resistance, from a population in which most cells contain three TopoII alpha gene copies (GLC4) to a population in which most cells contain only two copies. A partial revertant of the most resistant cell line displayed a shift back to the original situation. We conclude that the TopoII alpha gene copy number decrease per cell line is in good agreement with the decreased TopoII alpha mRNA and protein levels, and TopoII activity levels in these cell lines which were described previously.

The effect of sperm-mucus interaction test on the outcome of in vitro fertilization and ovulation induction combined with intrauterin insemination.

OBJECTIVE: To investigate the effect of sperm mucus penetration tests (SPT) on the fertilization rate (FR) and pregnancy rate (PR) in patients treated with either in vitro fertilization (IVF) or ovulation induction combined with intrauterine insemination (OI + IUI). METHODS: Retrospective analysis of a regional Infertility Unit database. Infertile couples where the women had normal ovarian function and a normal pelvis at laparoscopy and her partner had normal seminology who had failed at least two SPTs who were treated with either IVF or OI + IUI. These patients were compared with similar couples in whom SPTs were satisfactory (SPT + ve). Group A (SPT + ve) consisted of 46 patients who underwent 78 treatment cycles of IVF and Group B (SPT - ve) comprised 31 patients who underwent 35 IVF cycles. Group C (SPT + ve) consisted of 39 patients who underwent 84 treatment cycles with OI + IUI, and Group D (SPT - ve) consisted of 15 patients who underwent 37 cycles with the same treatment. RESULTS: In patients treated with IVF, the FR and PR per embryo transfer were 77.0% and 20.0%, respectively in Group A, and 64.0% and 22.6%, respectively in Group B. The differences in FRs were statistically significant (p < 0.001) but there was no difference in the PRs. In patients treated with OI + IUI, the PR per cycle were 22.0% in Group C and 16.2% in Group D. CONCLUSION: The results indicate that SPT failure was associated with a lower FR in IVF but this did not affect the PRs. Similarly there was no difference in PRs following OI + IUI.

A detailed study of the effect of videoframe rates of 25, 30 and 60 Hertz on human sperm movement characteristics.

A comparison was made of the movement characteristics of human spermatozoa analysed at three videoframe rates (25, 30 and 60 Hz) using two computerized motility analysers from Hamilton-Thorn Research (the HTM-2030 and the IVOS) operating at 25 and 30 Hz respectively. Analysis at 30 and 60 Hz was performed on the IVOS. The use of uncapacitated, capacitated and pentoxifylline-stimulated spermatozoa ensured a full range of movement characteristics was analysed. The velocity parameters curvilinear velocity and average path velocity were highly frame-rate dependent, and mean values increased with videoframe rate. An interaction of framing rate and time of data collection resulted in an increase in straight-line velocity with framing rate. Mean lateral head displacement and linearity were similar at 25 or 30 Hz but significantly depressed at 60 Hz. Beat-cross frequency increased by 74% when analysed at 60 rather than 30 Hz. The following criteria: curvilinear velocity > 100 microns/s, linearity < 65% and lateral head displacement > 7.5 microns, were used to define hyperactivated spermatozoa. Significantly more hyperactivated cells were identified at 30 Hz than 25 Hz (1-10%) but not at 60 Hz. A different population of cells is likely to have been identified as hyperactivated at 60 Hz due to alterations in component movement parameters from which the definition of hyperactivation was derived. In conclusion, direct comparisons should not be drawn between data analysed at 25 and 30 Hz. Analysis at 60 Hz introduced complex alterations which made simple comparisons with 30 Hz data invalid.

Spontaneous follicular and luteal function in infertile women with oligomenorrhoea: role of luteinizing hormone.

OBJECTIVE: There is a paucity of longitudinal endocrine studies of infertile patients with oligomenorrhoea. We have assessed the frequency and quality of spontaneous follicular development and luteal function in patients with oligomenorrhoea and infertility (PCOS), and have related the observed criteria to circulating LH activity. DESIGN: Prospective detailed investigations in a cohort of unselected patients. PATIENTS: Infertile women with oligomenorrhoea (PCOS, n = 131) presenting to the infertility clinic at the Royal Infirmary, Glasgow. MEASUREMENTS: Patients were monitored with frequent plasma oestradiol (E2) concentration assessments over a minimum period of 3 weeks, starting more than 2 weeks after a menstrual bleed. When follicular maturation was identified the patient provided daily blood samples through to her ensuing menstrual bleed, and E2, progesterone, total testosterone, FSH and LH were assessed in these samples. Luteal phase progesterone profiles were assessed between the days LH surge +2 and LH surge +6 by means of a progesterone index. RESULTS: Forty-eight per cent of the patients showed evidence of follicular development. The oestradiol profiles in the patients showing follicular growth were normal, but the progesterone curve was sub-normal in the early luteal phase, due to a high proportion of deficient luteal phases. The mean LH concentrations were elevated in the whole group, but no difference was observed between the mean LH values for those patients showing spontaneous follicular development and those who did not, and the incidence of ovulation was similar in the normal LH and elevated LH groups. Similarly, no relation was established between LH and the quantitative assessment of luteal phase progesterone profiles (progesterone index), and the distribution of progesterone indices was similar in the normal LH and elevated LH groups. Testosterone concentrations were positively correlated with LH (p = 0.008) but not with the incidence of spontaneous follicular growth. There was no significant difference in the incidence of spontaneous ovulation between the patients with elevated or normal mean follicular phase testosterone concentrations. CONCLUSION: The data indicate that both LH and testosterone secretion in PCOS were closely linked, but that neither was directly linked to the incidence or inhibition of spontaneous follicular development in PCOS, or to the disturbance in luteal phase progesterone profiles.