Quality of placental RNA: Effects of explant size and culture duration.

We evaluated the impact of placental micro (≤50 mg) and macro (∼200 mg) explants, oxygen concentration and culture method on placental RNA quality after long-term culture. Our findings show that micro explants cultured at 8% oxygen have the best RNA quality and tissue structure. Macro explants were less viable after long-term culture. Macro explants and explants undergoing syncytial degeneration produced poor quality RNA and should be avoided.

Comparison of Normal and Pre-Eclamptic Placental Gene Expression: A Systematic Review with Meta-Analysis.

Pre-eclampsia (PE) is a serious multi-factorial disorder of human pregnancy. It is associated with changes in the expression of placental genes. Recent transcription profiling of placental genes with microarray analyses have offered better opportunities to define the molecular pathology of this disorder. However, the extent to which placental gene expression changes in PE is not fully understood. We conducted a systematic review of published PE and normal pregnancy (NP) control placental RNA microarrays to describe the similarities and differences between NP and PE placental gene expression, and examined how these differences could contribute to the molecular pathology of the disease. A total of 167 microarray samples were available for meta-analysis. We found the expression pattern of one group of genes was the same in PE and NP. The review also identified a set of genes (PE unique genes) including a subset, that were significantly (p < 0.05) down-regulated in pre-eclamptic placentae only. Using class prediction analysis, we further identified the expression of 88 genes that were highly associated with PE (p < 0.05), 10 of which (LEP, HTRA4, SPAG4, LHB, TREM1, FSTL3, CGB, INHA, PROCR, and LTF) were significant at p < 0.001. Our review also suggested that about 30% of genes currently being investigated as possibly of importance in PE placenta were not consistently and significantly affected in the PE placentae. We recommend further work to confirm the roles of the PE unique and associated genes, currently not being investigated in the molecular pathology of the disease.

The role of coping strategies in predicting change in parenting efficacy and depressive symptoms among mothers of adolescents with developmental disabilities.

BACKGROUND: Parents of children with developmental disabilities (DD) face greater caregiving demands than parents of children without DD. There is considerable variability in parents' adjustment to raising a child with DD, however. In line with a strengths-based approach, this study explores coping strategies as potential mechanisms of resilience among mothers of adolescents with DD. This study examines the frequency with which mothers use various coping strategies and the extent to which those strategies moderate the relationship between adolescent behaviour problems and aspects of maternal well-being. Both positive and negative dimensions of well-being are explored, with maternal depressive symptoms and perceived parenting efficacy examined as outcomes cross-sectionally and longitudinally. METHODS: The present study focuses on 92 mothers and their adolescents with DD. The adolescents had a wide range of diagnoses, all with continuing special needs. Data were collected from mothers through interviews and self-administered questionnaires when their adolescents were aged 15 and aged 18. A structured assessment of the adolescent was completed during home visits at age 15. RESULTS: Mothers reported frequently using strategies of denial and planning but rarely using strategies of mental and behavioural disengagement to cope with recent stressful situations. Adolescent behaviour problems were found to contribute to greater symptoms of depression and lower feelings of parenting efficacy as well as increases in depressive symptoms over time. Mothers of sons, but not daughters, reported increases in parenting efficacy across their child's adolescent period. Above and beyond adolescent factors, several coping strategies emerged as significant predictors of mothers' symptoms of depression and perceived parenting efficacy. Moreover, use of Active Coping/Planning, Positive Reinterpretation/Growth, and Behavioural/Mental Disengagement as coping strategies moderated the impact of adolescent behaviour problems on maternal depressive symptoms. CONCLUSIONS: This study extends previous findings by focusing on both positive and negative dimensions of parent well-being during their child's adolescent period. Adolescence can be a stressful time for parents, with typical developmental tasks entailing additional strains for parents of adolescents with DD. The present findings point to several coping strategies that may reduce the impact of challenging behaviours during this period on mothers' symptoms of depression and feelings of parenting efficacy. Certain coping strategies were found to exert a greater impact on maternal well-being for parents of adolescents with higher levels of behaviour problems, suggesting that interventions may benefit from an increased focus on this group of mothers with heightened caregiving demands.

Investigation of DNA polymorphisms in SMAD genes for genetic predisposition to diabetic nephropathy in patients with type 1 diabetes mellitus.

AIMS/HYPOTHESIS: SMAD proteins are involved in multiple signalling pathways and are key modulators of gene expression. We hypothesised that genetic variation in selected SMAD genes contributes to susceptibility to diabetic nephropathy. METHODS: We selected 13 haplotype tag (ht) single nucleotide polymorphisms (SNPs) from 67 variants identified by resequencing the SMAD2 and SMAD3 genes. For SMAD1, SMAD4 and SMAD5 genes, genotype data were downloaded for 217 SNPs from Phase II of the International HapMap project. Of these, 85 SNPs met our inclusion criteria, resulting in the selection of 13 tag SNPs for further investigation. A case-control approach was employed, using 267 nephropathic patients and 442 controls with type 1 diabetes from Ireland. Two further populations (totalling 1,407 patients, 2,238 controls) were genotyped to validate initial findings. Genotyping was conducted using iPLEX, TaqMan and gel electrophoresis. RESULTS: The distribution of genotypes was in Hardy-Weinberg equilibrium. Analysis by the chi(2) test of genotype and allele frequencies in patients versus controls in the Irish population (n = 709) revealed evidence for the association of one allele at 5% level of significance (rs10515478, p(uncorrected) = 0.006; p(corrected) = 0.04). This finding represents a relatively small difference in allele frequency of 6.4% in the patient group compared with 10.7% in the control group; this difference was not supported in subsequent investigations using DNA from European individuals with similar phenotypic characteristics. CONCLUSIONS/INTERPRETATION: We selected an appropriate subset of variants for the investigation of common genetic risk factors and assessed SMAD1 to SMAD5 genes for association with diabetic nephropathy. We conclude that common polymorphisms in these genes do not strongly influence genetic susceptibility to diabetic nephropathy in white individuals with type 1 diabetes mellitus.

Use of an electronic nose to diagnose Mycobacterium bovis infection in badgers and cattle.

It is estimated that more than 50 million cattle are infected with Mycobacterium bovis worldwide, resulting in severe economic losses. Current diagnosis of tuberculosis (TB) in cattle relies on tuberculin skin testing, and when combined with the slaughter of test-positive animals, it has significantly reduced the incidence of bovine TB. The failure to eradicate bovine TB in Great Britain has been attributed in part to a reservoir of the infection in badgers (Meles meles). Accurate and reliable diagnosis of infection is the cornerstone of TB control. Bacteriological diagnosis has these characteristics, but only with samples collected postmortem. Unlike significant wild animal reservoirs of M. bovis that are considered pests in other countries, such as the brushtail possum (Trichosurus vulpecula) in New Zealand, the badger and its sett are protected under United Kingdom legislation (The Protection of Badgers Act 1992). Therefore, an accurate in vitro test for badgers is needed urgently to determine the extent of the reservoir of infection cheaply and without destroying badgers. For cattle, a rapid on-farm test to complement the existing tests (the skin test and gamma interferon assay) would be highly desirable. To this end, we have investigated the potential of an electronic nose (EN) to diagnose infection of cattle or badgers with M. bovis, using a serum sample. Samples were obtained from both experimentally infected badgers and cattle, as well as naturally infected badgers. Without exception, the EN was able to discriminate infected animals from controls as early as 3 weeks after infection with M. bovis, the earliest time point examined postchallenge. The EN approach described here is a straightforward alternative to conventional methods of TB diagnosis, and it offers considerable potential as a sensitive, rapid, and cost-effective means of diagnosing M. bovis infection in cattle and badgers.

A study comparing various noninvasive methods of detecting bladder cancer in urine.

OBJECTIVES: To compare the nuclear matrix protein (NMP)-22 assay, bladder tumour specific antigen (BTAstat) test, telomerase activity (using the telomeric repeat amplification protocol assay, TRAP) and a haemoglobin dipstick test for their ability to replace voided urine cytology (VUC) for detecting bladder cancer. PATIENTS AND METHODS: The study included 120 urological patients prospectively recruited and assessed before surgery. A single freshly voided urine sample (approximate 100 mL) was collected from each patient and aliquoted for each test. All assays were conducted according to the manufactures' guidelines; 79 patients were tested for telomerase activity. The results were then compared with VUC and the diagnosis confirmed by cystoscopy and histology. RESULTS: Fifty-two patients had histologically confirmed transitional cell carcinoma. The overall sensitivity for BTAstat, NMP22, telomerase, VUC and dipstick testing was 63%, 81%, 84%, 48% and 50%, respectively. Combining the results for telomerase and NMP22 gave a sensitivity of 100%. For G1 tumours the respective sensitivities were 23%, 62%, 56%, 23% and 15%, for G2 tumours, 68%, 86%, 92%, 50% and 41% and for G3 tumours 88%, 88%, 100%, 71% and 82%. For pTa tumours the respective detection rates were 48%, 70%, 84%, 39% and 30%, for pT1 tumours 80%, 90%, 90%, 50% and 50%, for pT2/pTis tumours, 100/100%, 100/100%, 100/100%, 88/100% and 88/83%. The overall specificity for the respective tests was 82%, 87%, 93%, 87% and 54%; combining the results of NMP22 and telomerase activity increased the specificity to 96%. CONCLUSIONS: There was significantly better detection than VUC when using the NMP22 and TRAP assay, especially for well-differentiated (P < 0.001 and 0.0027, respectively) and superficial tumours (P < 0.001 and 0.034, respectively). Combining the results of NMP22 and telomerase activity yielded values comparable with cystoscopy.

Standardisation of 11C.

The increasing use of positron emission tomography for medical imaging and the availability of short-lived positron emitters has raised concerns about the accuracy of calibration of secondary standard measurement systems and the viability of using a single long-lived positron emitter as a reference calibration source for all positron emitters. Potential problems arise because the 511 keV quanta arising from positron annihilation are not generally produced at the same point as the original disintegration. In addition, the secondary standard may also be responsive to the associated bremsstrahlung radiation. The magnitude of both effects depends on the positron end-point energy. In order to resolve these problems, it is necessary to produce absolute standards of these positron-emitting radionuclides and the work presented here details the results of such work with 11C.

Expression of the p53 homologue p63alpha and DeltaNp63alpha in the neoplastic sequence of Barrett's oesophagus: correlation with morphology and p53 protein.

BACKGROUND: While loss of p53 function is a key oncogenic step in human tumorigenesis, mutations of p53 are generally viewed as late events in the metaplasia-dysplasia-adenocarcinoma sequence of Barrett's oesophagus. Recent reports of a series of genes (p63, p73, and others) exhibiting close homology to p53 raise the possibility that abnormalities of these p53 family members may exert their influence earlier in the sequence. AIM: Following recent characterisation of expression of p63 and a major isoform DeltaNp63 by generation of an antiserum that recognises p63 isoforms, but not p53, our aim was a comparative study of expression of p63 protein and p53 protein in a morphologically well defined biopsy series representative of all stages of the metaplasia-dysplasia-carcinoma sequence in Barrett's oesophagus. METHODS: A series of 60 biopsy cases representing normal oesophagus through to invasive adenocarcinoma were stained, using immunohistochemistry, with antibodies to p63 and p53. All biopsies derived from patients with endoscopic and histopathological substantiation of a diagnosis of traditional/classical Barrett's oesophagus. RESULTS: There was exact concordance in p53 and p63 expression in more advanced forms of neoplasia, high grade dysplasia, and invasive adenocarcinoma, while p63, but not p53, was detected in the proliferative compartment of some non-neoplastic oesophageal tissue, in both squamous mucosa and in the non-neoplastic metaplastic glandular epithelium. CONCLUSIONS: In neoplastic Barrett's oesophagus there is upregulation of both p63 and p53 while p63 isoforms may well have an important role in epithelial biology in both non-metaplastic and metaplastic mucosa of the oesophagus. While abnormalities of p53 function represent an indisputable and critical element of neoplastic transformation, other closely linked genes and their proteins have a role in both the physiology and pathophysiology of the oesophageal mucosa.

The early detection and diagnosis of bladder cancer: a critical review of the options.

Bladder cancer has a high worldwide incidence matched by a tendency to recur, necessitating close and regular follow-up. Current methods of investigation of bladder cancer involve cystoscopy, ultrasound scanning and contrast urography, with additional information provided by cytology. These methods, although having a high detection rate, are expensive, time-consuming, invasive and uncomfortable. There is, therefore, a need for an inexpensive, noninvasive, quick and simple investigation with a high sensitivity and specificity for the detection of bladder cancer. There are an increasing number of molecular assays available for the detection of bladder cancer. From bladder tumour antigens to nuclear matrix proteins to adhesion molecules, cytoskeletal proteins and growth factors, urology has looked at them all to support the early detection and diagnosis of bladder cancer. This review critically discusses both the commercial as well as the research-based diagnostic assays available (their mode of action, overall accuracy - both by stage and grade, and their uses and limitations from both a clinical as well as a practical point of view). Aiming to give an insight into the options currently available for noninvasive bladder cancer diagnosis, it also provides prospective comment on what new methods/technologies may be useful in the medium term.

Noninvasive diagnosis of bladder carcinoma by enzyme-linked immunosorbent assay detection of CD44 isoforms in exfoliated urothelia.

The expression of variant isoforms of the adhesion molecule CD44 is correlated with the onset of neoplasia in many carcinomas. We have previously shown that noninvasive detection of bladder carcinoma is possible by analysis of anomalous CD44 expression in exfoliated urothelia. Although the sensitivity and specificity values obtained for the detection of bladder tumors using RT-PCR and Western blotting methods were superior to those obtained using urine cytology, the application of such techniques is inconvenient for routine diagnostic use. We now report the design and development of a sandwich-ELISA system for the reliable detection of CD44 protein extracted from sedimented urothelial cells in voided urine. Naturally micturated urine samples were obtained from 53 patients with newly diagnosed bladder cancer and from 65 subjects with no evidence of disease; patients with gross hematuria were excluded because of interference with the assay. To demonstrate the diagnostic potential of the system, a "gate" was imposed at N (max), i.e., the highest absorbance value obtained from a sample known to be tumor free. All values above this value were assumed to be indicative of the presence of a tumor. Using this parameter, 42 of 53 (81.1%) patients with histologically confirmed bladder tumors were correctly diagnosed. Correspondingly, under these conditions, the assay is 100% specific for tumor detection, with a sensitivity of 81.1%, which equates to a positive predictive value of 100% and a negative predictive value of 81.1%. A further 54 patients who had previously received treatment for bladder cancer but were currently clinically disease-free were also investigated. Of these, 47 of 54 (87%) were correctly diagnosed to be tumor-free, which in this group equates to a positive predictive value of 87% and a negative predictive value of 100%. The data presented demonstrate that the rapid and accurate detection of elevated levels of CD44 protein isoforms in exfoliated urothelial cells is applicable to the identification and monitoring of primary and recurrent bladder cancer.

Telomerase activity in soft-tissue and bone sarcomas.

The telomerase enzyme is a reverse transcriptase capable of replacing the telomeric DNA sequences that are lost at each cell division. Telomerase activation permits extended cell proliferation beyond normal senescence checkpoints, and accordingly, telomerase activity has been detected in a wide range of malignant cells and tissues but is absent in terminally differentiated somatic cells. To date, the majority of cancer-related telomerase analyses have been performed on carcinomas that originate from epithelial cells, and few reports have included tumors originating from nonepithelial cells. In this study, we used the PCR-based telomeric repeat amplification protocol (TRAP) to assay telomerase activity in nuclear protein extracts obtained from a range of malignant and benign connective tissue lesions. In total, 62 histologically diagnosed specimens were analyzed including 37 sarcomas, 7 benign mesenchymal tumors, 12 normal tissue samples, and 6 carcinoma metastases obtained from bone. Thirty (81%) of the 37 primary sarcoma samples contained telomerase activity, and four of the six carcinoma metastases were also positive. Conversely, telomerase activity was detectable in only one of seven benign lesions and in none of the 12 normal connective tissue controls. Tumors of connective tissue origin can sometimes be difficult to categorize and to evaluate microscopically with regard to clinical management. As is the case in carcinomas, the presence of telomerase activity appears to be indicative of malignancy in mesenchymal tumor biopsy material and therefore may be useful as an adjunct to the pathologist in the assessment of borderline cases.

CD44 expression in cervical intraepithelial neoplasia (CIN) and carcinoma.

BACKGROUND: The expression of the CD44 gene is markedly changed in many neoplastic tissues. The identification of tumor-specific CD44 expression patterns may aid tumor diagnosis. METHODS AND RESULTS: The transcription and translation of the CD44 gene were analyzed by reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization and by immunohistochemistry. Samples were obtained from 24 normal and 24 neoplastic or malignant human cervical tissues. Southern blot hybridization analysis of RT-PCR products revealed an increase in the size and number of CD44 standard and variant transcripts in malignant cervical tissues compared with corresponding normal tissues. Misprocessing of mRNA was indicated in cervical carcinoma cells by the retention of intronic sequences. Multiple CD44 mRNA and protein isoforms were present throughout carcinoma tissues, whereas localization was restricted to the basal epithelium in normal cervical tissue. Analysis of desquamated cervical cells from cases of cervical intraepithelial neoplasia stages I-III showed progressively deranged patterns of CD44 expression, with more alterations being detected in the more advanced stages. CONCLUSIONS: Marked alterations in CD44 expression occur in cervical tissues during progression to malignancy. CD44 expression analysis could aid the early diagnosis of cervical malignancy using minimally invasive methods.

Who wants to be a specialist?

The UK has not developed the degree of limited practice which is common overseas and is now growing in Europe. However, changes in European legislation have brought about a recognition of these specialist disciplines within dentistry.

Rapid analysis of distinctive CD44 RNA splicing preferences that characterize colonic tumors.

In normal tissues, the steady-state level of CD44 mRNA is low, and the variety of alternatively spliced transcripts produced by this complex gene is limited. Conversely, increased and disorderly expression of this gene has been observed in a number of types of cancer. This study analyzed the order in which the CD44 variant exons are spliced together in gastrointestinal tumor cell lines and in 20 colonic carcinomas and matched normal mucosa. We used a PCR-based assay to analyze specific exon junctions at the boundary of the standard and variant regions of the CD44 gene transcripts. This revealed characteristically different splicing preferences in colonic tumor and normal tissues. The junction of exon 5 to exon 8 appeared to be the most prevalent in normal mucosa, whereas the presence of junctions between exon 5 and either exon 7, 9, or 11 were increased markedly in tumor samples. These observations demonstrate that the unusual variety of CD44 transcripts in cancer cells results from the fidelity of alternative splicing mechanisms being compromised and are potentially useful as tumor cell markers in diagnostic assays.

Nonparenchymal cells from regenerating rat liver generate interleukin-1alpha and -1beta: a mechanism of negative regulation of hepatocyte proliferation.

Following experimental partial hepatectomy of 70% in the rat, there is a semisynchronized surge of hepatocyte proliferation that ceases after 48 to 72 hours. Little is known about the determinants governing the termination of the proliferative phase, although transforming growth factor (TGF) beta has been implicated as an important inhibitor of hepatocyte replication in this model. We previously reported an additional non-TGF-beta inhibitor in medium conditioned by nonparenchymal cells isolated from regenerating liver (CM-NPC-Reg) between 24 and 48 hours after partial hepatectomy, but it was not found in medium conditioned by nonparenchymal cells from unoperated control liver. CM-NPC-Reg suppressed replicative DNA synthesis of primary rat hepatocytes in response to hepatocyte growth factor (HGF), epidermal growth factor (EGF), or TGF-alpha as assessed by 3H-thymidine incorporation. We now present evidence that interleukin (IL)-1 is the major inhibitor of hepatocyte DNA synthesis present in CM-NPC-Reg. IL-1 receptor antagonist abrogated the inhibition, as did antibodies to rat IL-1alpha and -beta; a combination of both antibodies was required, implicating both IL-1alpha and IL-1beta as active constituents in CM-NPC-Reg. To investigate in vivo changes in IL-1 expression, we assessed expression of IL-1alpha messenger RNA (mRNA) in whole rat liver following partial hepatectomy; mRNA was down-regulated at 10 hours in the pre-replicative phase of liver regeneration and up-regulated at 24 hours and 48 hours when proliferation is waning. Rat hepatocytes isolated from liver 24 hours after partial hepatectomy showed increased sensitivity to the inhibitory action of IL-1. Exogenous IL-1beta, administered parenterally to a group of rats at 0 and 12 hours after partial hepatectomy significantly reduced the incorporation of the thymidine analogue, bromodeoxyuridine (BrdU), into hepatocytes at 18 hours. These data indicate that nonparenchymal cells isolated from regenerating rat liver elaborate IL-1, and support the hypothesis that IL-1 plays a role suppressing hepatocyte proliferation and terminating the surge of DNA synthesis induced after partial hepatectomy.

Telomerase activity in bladder carcinoma and its implication for noninvasive diagnosis by detection of exfoliated cancer cells in urine.

BACKGROUND: Telomerase is an enzyme that can reconstitute the ends (telomeres) of chromosomes after cell division and thus circumvent the cumulative damage that occurs in normal adult somatic cells during successive mitotic cycles. Recently, it has been proposed that this enzyme should, therefore, be detectable in immortal malignant cells but not in their normal counterparts, which stop dividing and senesce. Accordingly, telomerase activity has been reported in many types of malignant tumors, including those of the gastrointestinal tract, breast, and lung but little information was available regarding its status in bladder carcinoma or in exfoliated cancer cells. METHODS: In the current study, telomerase activity was examined by a polymerase chain reaction-based assay designated TRAP (telomeric repeat amplification protocol) in tissue samples from 56 bladder carcinomas, 17 nonneoplastic bladder lesions, and 2 dysplastic lesions of the urinary tract. The feasibility of identifying cancer patients by the detection of telomerase activity in exfoliated cancer cells in the urine was also investigated. Such activity was assayed in centrifuged urine cell pellets from 26 bladder carcinoma patients and from 83 patients with no evidence of malignant disease. RESULTS: Evidence of telomerase was detected in solid tissue specimens from 48 of the 56 bladder carcinomas (86%) regardless of tumor stage or differentiation, whereas it was not found in any normal bladder tissue specimen. However, it was present in the dysplastic bladder lesions as well as in nearly all Stage I well differentiated carcinomas, suggesting that its activation occurs for the early stages of carcinogenesis and could perhaps be a useful marker for the detection of early primary or recurrent bladder tumors. Telomerase activity was detected with various signal intensities in urine specimens from 16 of the 26 patients with bladder carcinoma (62% sensitivity), whereas only 3 of 83 nonmalignant urine samples showed any activity (96.4% specificity); this was very weak. CONCLUSIONS: These results suggest that telomerase could be a good diagnostic marker for the early noninvasive identification of patients with bladder carcinoma by facilitating the detection of exfoliated immortal cancer cells in their urine.