Validation of the pedigree assessment tool (PAT) in families with BRCA1 and BRCA2 mutations.

BACKGROUND: The lifetime risk of breast cancer (BC) in patients with hereditary breast cancer syndromes is as high as 80%. The Pedigree Assessment Tool (PAT) is a scoring system to aid in identifying these patients. This validation study compares the PAT to BRCA gene mutation probability models in predicting suitability for genetic referral. METHODS: Retrospective review identified subjects undergoing genetic counseling and BRCA testing from 2001 to 2008 at two institutions. PAT score and BRCA mutation probabilities were calculated using Myriad II and Penn II models. Comparisons were made between models in ability to discriminate patients appropriate for genetic evaluation based on accuracy in predicting a positive test result. RESULTS: Records evaluated represent 520 families. BRCA testing revealed 146 mutation-positive and 374 mutation-negative families. c-Statistic analysis was used to compare the discriminating ability of the models to correctly assign families as mutation (+) and (-). Both the PAT and Penn II model outperformed the Myriad II model. Using a threshold PAT score >or=8 and mutation probability >or=10% to assign families as mutation (+) versus (-), sensitivity, specificity, and positive and negative predictive values were calculated for each model. The PAT was more sensitive than the Myriad II model and more specific than the Penn II model. CONCLUSIONS: In overall performance, the PAT is at least comparable to the Myriad II and Penn II models in screening women appropriate for genetic referral. Simplicity and identification of families with non-BRCA hereditary BC syndromes suggest that the PAT is better suited for BC risk screening.

ERK1 and ERK2 activation by chemotactic factors in human eosinophils is interleukin 5-dependent and contributes to leukotriene C(4) biosynthesis.

Eosinophils, the major immune effector cells contributing to allergic inflammation and asthma, are profoundly affected by interleukin (IL) 5 with respect to their differentiation, viability, recruitment, and cytotoxic effector functions. IL-5 enhances eosinophil responsiveness to a variety of chemotactic factors via a process called priming, although the molecular mechanism is unknown. In this study, we report that, following IL-5 priming of eosinophils, chemotactic agents including fMet-Leu-Phe, IL-8, and RANTES, promote vigorous transient activation of ERK1 and ERK2. In contrast, these chemotactic factors stimulate weak or indiscernible ERK activation in unprimed eosinophils. Furthermore, this intracellular marker of priming is selective for IL-5-related cytokines, in that it is observed following exposure to IL-5 and granulocyte macrophage-colony stimulating factor but not to interferon-gamma, stem cell factor, tumor necrosis factor alpha, or IL-4. Interestingly, priming of chemoattractant-induced ERK activation is accompanied by an increase in association of tyrosine-phosphorylated proteins with the adapter protein Grb2. The biological relevance of ERK activation to IL-5 priming is supported by the observation that inhibition of ERK activity by treatment with the MEK inhibitors PD98059 or U0126 inhibited the release of leukotriene C(4) stimulated by fMet-Leu-Phe in IL-5-primed eosinophils. These data provide evidence for a previously undescribed fundamental mechanism by which stimulation of IL-5 family receptors induces a rapid phenotypic alteration in the signal transduction pathways of chemotactic receptors, enabling their activation of the ERK1 and ERK2 pathway and contributing to the capacity of these cells to synthesize LTC(4).

Expression and secretion of TGF-beta isoforms and expression of TGF-beta-receptors I, II and III in normal and neoplastic human breast.

We investigated gene expression of the TGF-beta signalling system (including peptides and receptors) in normal and malignant breast tissue. Additionally, gene and protein expression was determined in a series of primary epithelial and stromal cultures derived from these tissues. TGF-beta isoforms and their receptors were expressed by both tissue sets, however the percentage of samples expressing each transcript varied. In normal breast, both TGF-beta1 and TGF-beta3 were found in most samples (88 and 89% respectively), with fewer expressing TGF-beta2 (68%). A similar pattern was evident in the tumours. Type I receptor of TGF-beta was constitutively expressed in normal breast and observed in most tumours (90%). Type II and III receptors of TGF-beta were expressed less frequently, although the type II receptor was mainly expressed by tumours (P=0. 0075). All primary cultures produced TGF-beta1 and TGF-beta2. Comparing respective cell populations, tumour stromal cells produced significantly more TGF-beta1 than those derived from normal breast (P<0.0001). Linear regression analysis showed stromal cultures derived from breast tumours exhibited a strong positive correlation (r=0.976) in the production of TGF-beta1 and TGF-beta2. Thus, TGF-beta and TGF-beta-receptors are widely and differentially expressed by normal and malignant breast and secretion of this peptide by epithelial and stromal cultures, in particular those derived from tumours, confirms its potential as an autocrine/paracrine regulator in breast cancer.

17Beta-hydroxysteroid dehydrogenase type 1, 2, 3, and 4 expression and enzyme activity in human anterior pituitary adenomas.

17Beta-hydroxysteroid dehydrogenase (17betaHSD) isoforms reversibly catalyze the final step in the formation of estradiol (E2) from estrone (E1) and the formation of testosterone from androstenedione. We have investigated 17betaHSD type 1, 2, 3, and 4 gene expression and 17betaHSD estrogenic activity in human anterior pituitary adenomas. 17BetaHSD messenger ribonucleic acid (mRNA) expression was studied by RT-PCR in 42 pituitary tumors and 3 normal pituitaries, 17betaHSD activity was studied in 11 tumors and 17betaHSD type 1 was immunolocalized in vitro in 6 tumors. 17BetaHSD type 1 gene expression was detected in 34 of 42 adenomas in all tumor subtypes; 17betaHSD type 2 mRNA was detected in 18 of 42 adenomas, but not in prolactinomas; 17betaHSD type 3 mRNA was detected in 12 of 42 adenomas, but not in corticotropinomas; 17betaHSD type 4 was expressed in 20 of 42 adenomas by all adenoma subtypes. Reversible 17betaHSD activity was found in 9 of 11 adenomas, and 17betaHSD type 1 immunopositivity was cytoplasmically distributed in all 6 adenomas in vitro. All 4 17betaHSD isoforms are variably expressed in human anterior pituitary adenomas, which also show 17betaHSD enzyme activity, suggesting that 17betaHSD may play an important role in regulating the local cellular levels of estradiol.

Expression of cytokine messenger RNA in normal and neoplastic human breast tissue: identification of interleukin-8 as a potential regulatory factor in breast tumours.

The presence of mRNA transcripts for cytokines in normal and neoplastic human breast tissue has been investigated. Using reverse transcriptase-linked polymerase chain reaction (RT-PCR), we have specifically screened for the following cytokines: interleukin (IL)-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, tumour necrosis factor (TNF)-alpha, TNF-beta and interferon (IFN)-gamma. No significant differences in expression of IL-1alpha, IL-1beta, IL-4, IL-6, TNF-alpha or TNF-beta were observed between the 2 groups of tissues. However, there was a significant difference in expression of IL-8 transcripts (p = 0.0017) which was higher in the neoplastic population. Transcripts for IL-2, IL-3, IL-5, IL-7 and IFN-gamma were not detected in either group. There was no evidence of associations between cytokine expression and tumour histological grade, patient age or lymph node metastases. Correlating tumour types with specific cytokine transcripts revealed high expression of IL-8, and to a lesser extent, IL-8 and TNF-beta irrespective of tumour origin. Analysis of primary epithelial and stromal cultures derived from both types of tissue showed that increased levels of IL-8, but not IL-6, were secreted by cells obtained from tumours. Thus, breast tissue of both normal and neoplastic origin expresses a wide range of cytokines. Increased or aberrant expression of cytokines, in particular IL-8, may be involved in the development/progression of breast cancer.

Identification of proliferating human anterior pituitary adenoma cells in vitro.

A method to determine whether dispersed human anterior pituitary adenoma cells proliferate in mixed culture was developed. Fifteen pituitary adenomas were dispersed enzymatically to single cells, following which twelve were double immunostained after eight days. Proliferating cells were identified immunologically following one hour of bromo-deoxyuridine incorporation. Adenoma cells were subsequently identified with an anti-neuron-specific enolase antibody system. A time course of bromo-deoxyuridine labelling was performed on three nonfunctional adenomas over a four day period, with bromo-deoxyuridine being added to cultures at one hour, 24 hours and four days prior to immunostaining. Double immunolabelled cells were unambiguously identified by a dark brown nucleus surrounded by red cytoplasm. Eight out of 12 pituitary adenomas (two prolactinomas, three nonfunctional, three growth hormone secreting) showed an increased bromo-deoxyuridine labelling index (range 0.1%-1.4%). Bromo-deoxyuridine incorporation over four days showed an increase in bromo-deoxyuridine from 0.02%, 0.03% and 3.3% at one hour to 10.1%, 1.3% and 5.0% at four days, respectively, but evidence of mitosis was scant. This study shows that pituitary adenomas may proliferate in vitro and that this double immunostaining method may be used as an in vitro proliferation assay in a mixed cell population.

A comparison of proliferation indices in human anterior pituitary adenomas using formalin-fixed tissue and in vitro cell culture.

The authors compared detection methods for cell proliferation in human anterior pituitary adenomas using histological sections and dispersed cell culture. After tumor cells had been grown for 4 days in dispersed culture, bromodeoxyuridine (BUdR), proliferating cell nuclear antigen (PCNA), and Ki-67 were compared by double immunostaining and contrasted with single staining of PCNA and Ki-67 indices in the corresponding histological sections from 12 human pituitary adenomas. In vitro, the BUdR labeling index was positive in six of 12 tumors (range < 0.1-5.1%), 10 of 12 tumors were PCNA-positive (range < 0.1-100%), and Ki-67 was positive in 10 of 12 adenomas (range < 0.1-8%). In vitro, BUdR and Ki-67 gave similar proliferative indices for 10 of 12 adenomas. In vivo, the PCNA labeling index was positive in 12 of 12 adenomas (range 0.9-95%) and Ki-67 was positive in 11 of 12 adenomas (range < 0.1-2%). Tumors with a labeling index less than 0.1% were considered to be negative for proliferation. High PCNA values were found in vitro and in vivo, whereas Ki-67 labeling indices were similar in vitro and in vivo for nine of 12 adenomas. It is concluded that Ki-67 proliferative indices in vivo reflect those found in vitro, at least after 4 days in dispersed culture, but that PCNA overestimates pituitary adenoma proliferation in histological sections as well as in dispersed culture.

Apoptosis and p53 suppressor gene protein expression in human anterior pituitary adenomas.

Human anterior pituitary adenomas proliferate and express the p53 tumour suppressor gene protein, but it is not known if apoptosis (programmed cell death) occurs. Therefore, the detection of apoptosis was undertaken in tumorous human anterior pituitary tissue and compared with p53 protein expression, tumour type and tumour size. Apoptosis (detected by the in situ end labelling technique) and p53 suppressor gene protein (detected by DO.1-antibody immunocytochemistry) were determined in formalin-fixed and paraffin-embedded tissue from 37 human pituitary adenomas (2 macroprolactinomas, 9 somatotrophinomas and 26 non-functioning adenomas). Two normal anterior pituitaries were also included in this study. Pre-operative tumour size was scored 1 to 4 from magnetic resonance imaging radiology. Apoptosis was found in 7 of 29 tumours (24%), 11% of somatotrophinomas and 33% of non-functioning adenomas, although this difference was not significant. The p53 tumour suppressor protein was found in 7 of 31 tumours (23%), 33% of somatotrophinomas and 19% of non-functioning adenomas. Apoptosis and p53 protein expression were not found in normal anterior pituitary. In conclusion, apoptosis occurs in human anterior pituitary adenomas, but no significant association was found between apoptosis and p53 protein expression, tumour type or tumour size.

Cytokine expression in human anterior pituitary adenomas.

OBJECTIVE: There is increasing evidence for the role of cytokines in pituitary differentiated function and tumorigenesis, but the spectrum of cytokines found in the pituitary is unknown. Therefore profiles of cytokine expression were determined in different human anterior pituitary adenoma sub-types. DESIGN: The reverse transcriptase-linked polymerase chain reaction (PCR) was used to identify the presence of cytokine mRNA within human pituitary adenomas. PATIENTS: Seventeen pituitary adenoma biopsies removed at transsphenoidal surgery were examined: 4 somatotrophinomas, 7 non-functional adenomas, 4 prolactinomas, one case of Cushing's disease and one case of Nelson's syndrome. MEASUREMENTS: RNA was extracted from each adenoma biopsy and reverse transcribed into cDNA. This was specifically amplified in a PCR using oligonucleotide primers complementary to each cytokine. The cytokines investigated were interleukin (IL)-I alpha, IL-I beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, tumour necrosis factor (TNF)-alpha, TNF-beta and transforming growth factor (TGF)-beta 1, beta 2 and beta 3. The products of each PCR were visualized using agarose gel electrophoresis. RESULTS: All 17 adenomas expressed IL-8 transcripts, but no expression of IL-2, IL-5 or IL-7 was found. IL-6 was expressed in all 4 somatotrophinomas, 3 of 7 non-functional tumours, 2 of 4 prolactinomas and in the single case of Nelson's syndrome. At least one of the 3 isoforms of TGF-beta was found in all but 2 tumours; one prolactinoma and one non-functional adenoma. IL-1 alpha, IL-beta, IL-4, TNF-alpha and TNF-beta were expressed sporadically by individual adenomas. CONCLUSION: These data suggest that whilst IL-8 may be important, the local expression of the cytokines IL-2, IL-5 and IL-7 is not important in human anterior pituitary tumorigenesis.

Expression of the transferrin receptor in human anterior pituitary adenomas is confined to gonadotrophinomas.

OBJECTIVE: The human pituitary gland is affected selectively by conditions associated with iron deposition, but the mechanisms for this are unknown. In this study we have determined whether the transferrin receptor, which mediates iron uptake by cells, could be detected immunocytochemically in human pituitary adenomas in vitro. PATIENTS: Data were derived from 35 patients undergoing transsphenoidal surgery and included 13 with clinically non-functioning adenomas, 15 with acromegaly, 4 prolactinomas, 2 patients with Cushing's disease and one patient with Nelson's syndrome. MEASUREMENTS: Transferrin receptor immunopositivity was determined for each adenoma in dispersed cell culture using a specific monoclonal antibody. RESULTS: Eight of 13 clinically functionless adenomas showed immunopositive transferrin receptor expression, whilst adenomas from 15 patients with acromegaly, 4 prolactinomas, 2 Cushing's syndrome and one patient with Nelson's syndrome were negative. The eight transferrin receptor positive tumours were gonadotrophinomas and accounted for eight of the nine tumours which secreted and immunostained for FSH; all eight also secreted and immunostained for LH. CONCLUSIONS: These findings may reflect a special requirement for iron by gonadotrophin secreting cells in comparison to other pituitary cell types and this could underlie the reasons why in the normal pituitary these cells are especially susceptible to malfunction in iron overload syndromes such as genetic haemochromatosis and beta-thalassaemia.

The resurrection and the life.

This first-person account of a woman undergoing manic-depression concurrent with alcohol and drug problems highlights the difficulties of attaining help from a system geared toward treating one or the other but not both, and hints at the positive outcomes possible when an integrated approach to co-occurring addictive and mental disorders--one that incorporates the knowledge and perspectives of the client and the family as well as the professional--is available.

Synthesis of nucleotides by phosphotransferase from human tissues.

Several human tissues (prostate greater than liver greater than kidney greater than spleen) contain phosphotransferase capable of synthesizing nucleoside monophosphate by low-energy phosphate transfer to pyrimidines, purines as well as ribo- and deoxyribonucleosides. Phenyl phosphate, AMP, TMP, GMP and IMP were good phosphate donors while thymidine, deoxyadenosine and deoxyuridine are the most effectively utilized substrates. The phosphatransferase activity is present in all particulate fractions with most activity in microsomes. The enzyme is being purified from human tissues.

A quantitative computerized recording method in clinical education: a pilot project.

This paper describes an approach to a quantitative computerized method for recording the clinical education experiences of physical therapy students. While the project was conducted in the field of physical therapy, it provides a model that can be adapted to other allied health fields. The objectives, methodology, outcome and future prospects of the project are discussed.