Gene expression profiles in HPV-infected head and neck cancer.

Epidemiological and laboratory evidence indicate that, in addition to tobacco and alcohol, human papillomaviruses (HPV) play an important aetiological role in a subset of head and neck squamous cell carcinoma (HNSCC). To evaluate the molecular pathogenesis of HPV-infected HNSCC, we compared gene expression patterns between HPV-positive and -negative HNSCC tumours using cDNA microarrays. Tumour tissue was collected from 42 histologically confirmed HNSCC patients from an inner-city area of New York. Total DNA and RNA were extracted and purified from frozen tumour samples and gene expression levels were compared to a universal human reference RNA standard using a 27 323 cDNA microarray chip. HPV detection and genotyping were performed using an MY09/11-PCR system and RT-PCR. HPV was detected in 29% of HNSCC tumours. Most harboured only HPV16 and expressed the HPV16-E6 oncogene. HPV prevalence was highest in pharyngeal tumours (45%). Gene expression patterns that differentiated HPV-positive from negative tumours were compared by supervised classification analysis, and a multiple-gene signature was found to predict HPV16 prevalence in primary HNSCC with a false discovery rate < 0.2. Focusing on never-smokers, we further identified a distinct subset of 123 genes that were specifically dysregulated in HPV16-positive HNSCC. Overexpressed genes in HPV-positive HNSCC tumours included the retinoblastoma-binding protein (p18), replication factor-C gene, and an E2F-dimerization partner transcription factor (TFDP2) that have also been found to be overexpressed in cervical cancer. An additional subset of genes involved in viral defence and immune response, including interleukins and interferon-induced proteins, was found to be down-regulated in HPV-positive tumours, supporting a characteristic and unique transcriptional profile in HPV-induced HNSCC.

Classification of DNA methylation patterns in tumor cell genomes using a CpG island microarray.

Our group has initiated experiments to epigenetically profile CpG island hypermethylation in genomic DNA from tissue specimens of head and neck squamous cell carcinoma (HNSCC) using a microarray of 12,288 CpG island clones. Our technique, known as a methylation-specific restriction enzyme (MSRE) analysis, is a variation of the differential methylation hybridization (DMH) technique, in that it is not an array comparison of two DNA samples using methylation-specific restriction enzymes. Instead, it is a comparison of a single DNA sample's response to a methylation-sensitive restriction enzyme (HpaII) and its corresponding methylation-insensitive isoschizomer (MspI). Estimation of the reproducibility of this microarray assay by intraclass correlation (ICC) demonstrated that in four replicate experiments for three tumor specimens, the ICC observed for a given tumor specimen ranged from 0.68 to 0.85 without filtering of data. Repeated assays achieved 87% concordance or greater for all tumors after filtering of array data by fluorescence intensity. We utilized hierarchical clustering on a population of 37 HNSCC samples to cluster tumor samples with similar DNA methylation profiles. Supervised learning techniques are now being utilized to allow us to identify associations between specific epigenetic signatures and clinical parameters. Such techniques will allow us to identify select groups of CpG island loci that could be used as epigenetic markers for both diagnosis and prognosis in HNSCC.

A role for schwann cells in the neuroregenerative effects of a non-immunosuppressive fk506 derivative, jnj460.

UNLABELLED: FK506 and its non-immunosuppressive derivatives represent a class of pharmacological agents referred to as immunophilin ligands that have been reported to promote neuroregeneration and survival in several experimental models; however their cellular and molecular mechanisms of action have not been well established. Here we characterize a new immunophilin ligand that interacts with both FK506 binding protein 12 (FKBP12) and FKBP52, and demonstrate that JNJ460 induces neurite outgrowth from freshly explanted dorsal root ganglia (DRG) in a Schwann cell-dependent manner. Purified cultures of neurons fail to respond to these drugs, but cultures containing Schwann cells and neurons respond with neurite outgrowth, as do neurons grown in conditioned medium from JNJ460-treated Schwann cells. Using microarray analysis and a transcription reporter assay, we show that JNJ460 induces a series of transcriptional changes that occur in a temporal cascade. Among the Schwann cell-expressed genes upregulated following JNJ460 treatment is the POU transcription factor SCIP, which has been shown to regulate Schwann cell gene transcription and differentiation. JNJ460 potentiated transforming growth factor beta (TGF-beta)-induced transcriptional activation and SCIP induction in Schwann cells, by altering the interaction between FKBP12 and the TGF-beta type I receptor, TbetaR1. Finally, to test whether JNJ460 enhances neurite regeneration in vivo, we treated animals with JNJ460 for 30 days following mechanical transection of the sciatic nerve and demonstrated myelin and axonal hypertrophy at the ultrastructural level. Collectively, these data suggest that Schwann cells play an important role in the biological effects of immunophilin ligands by affecting neuron-glial signaling during regeneration. SUMMARY: The cellular and molecular mechanisms responsible for the regenerative effects of immunophilin ligands are not well understood. Here we show that the neuritogenic effects of JNJ460 in a DRG model depend on interactions between neurons and Schwann cells. Treatment of purified Schwann cells with JNJ460 alters Schwann cell gene expression, and promotes the generation of factors that act on neurons. These data indicate that Schwann cells play an important role in the actions of immunophilin ligands.

Functional limitations of school-aged children seen in primary care.

OBJECTIVE: The purpose of the present study was to assess the prevalence of functional limitations in children seen in a large paediatric practice network and to identify sociodemographic, family and psychosocial factors related to functional limitations. STUDY DESIGN: Cross-sectional analysis. POPULATION: Children were recruited from two large, practice-based primary care research networks during their paediatric office visits. For the present study, participants included 14 630 school-aged children (ages 6-15 years) and their caregivers. OUTCOMES MEASURED: Parents completed written questionnaires including the Pediatric Symptom Checklist, the Family Apgar and the Functional Limitations Index. RESULTS: Findings indicated that 15% of children surveyed had some limitation in their daily functioning. More children had schoolwork and physical function limitations than limitations in personal and self-care. Logistic regression equations predicted functional limitations and health status in children from a model of sociodemographic factors, psychosocial symptoms and family functioning. CONCLUSIONS: A low but significant number of school-age children seen in the primary care setting experience functional limitations. Children with any psychosocial symptoms were at increased risk for functional limitations, indicating the critical need to screen for functional impairment in children with suspected behavioural or emotional problems. A screening tool of functional limitations may be useful for assessing the presence or absence of such limitations in children's daily function and warrants further investigation.

Development of gonadotropes may involve cyclic transdifferentiation of growth hormone cells.

The cyclic rise in expression of anterior pituitary gonadotropins coincides with the appearance of cells sharing gonadotropic and somatotropic phenotypes. To learn more about possible factors that regulate the origin of this cell type, we studied the time of appearance of cells that co-expressed growth hormone (GH) and gonadotropins and estrogen receptors during the estrous cycle and compared this timing with known changes in regulatory hormones or their receptors. The first event in this cell population is an increase in expression of estrogen receptor (ER)beta by GH cells from estrus to metestrus suggesting that estrogen may mediate this early change. Expression of GH mRNA rises rapidly from metestrus to mid-cycle. The rise is seen first in GH cells and then in cells with luteinizing hormone (LH) antigens. These data suggest that, early in the cycle, cells bearing GH and growth hormone releasing hormone (GHRH) receptors begin to produce LH and gonadotropin releasing hormone (GnRH) receptors. Early in proestrus, there is an increase in cells with GH and follicle-stimulating hormone (FSH) suggesting that this set of multipotential cells develops later than GH-LH cells. This fits with earlier studies showing the later rise in expression of FSH mRNA. Collectively these data suggest that the anterior pituitary contains a subset of GH cells that have the capacity to respond to multiple releasing hormones and support more than one system.

Control of gonadotropin secretion by follicle-stimulating hormone-releasing factor, luteinizing hormone-releasing hormone, and leptin.

Fractionation of hypothalamic extracts on a Sephadex G-25 column separates follicle-stimulating hormone-releasing factor (FSHRF) from luteinizing hormone-releasing hormone (LHRH). The FSH-releasing peak contained immunoreactive lamprey gonadotropin-releasing hormone (lGnRH) by radioimmunoassay, and its activity was inactivated by an antiserum specific to lGnRH. The identity of lGnRH-III with FSHRF is supported by studies with over 40 GnRH analogs that revealed that this is the sole analog with preferential FSH-releasing activity. Selective activity appears to require amino acids 5-8 of lGnRH-III. Chicken GnRH-II has slight selective FSH-releasing activity. Using a specific lGnRH-III antiserum, a population of lGnRH-III neurons was visualized in the dorsal and ventral preoptic area with axons projecting to the median eminence in areas shown previously to control FSH secretion based on lesion and stimulation studies. Some lGnRH-III neurons contained only this peptide, others also contained LHRH, and still others contained only LHRH. The differential pulsatile release of FSH and LH and their differential secretion at different times of the estrous cycle may be caused by differential secretion of FSHRF and LHRH. Both FSH and LHRH act by nitric oxide (NO) that generates cyclic guanosine monophosphate. lGnRH-III has very low affinity to the LHRH receptor. Biotinylated lGnRH-III (10(-9) M) labels 80% of FSH gonadotropes and is not displaced by LHRH, providing evidence for the existence of an FSHRF receptor. Leptin has equal potency as LHRH to release gonadotropins by NO. lGnRH-III specifically releases FSH, not only in rats but also in cows.

Boys starting school disadvantaged: implications from teachers' ratings of behaviour and achievement in the first two years.

BACKGROUND: Consistent evidence indicates that low socio-economic status (SES) acts as an important stressor and vulnerability factor for children's school learning. However, specific mechanism(s) of this process are still not well understood. AIM: This study was a follow-up of the classroom learning behaviour and perceived achievement of low and middle income children after two years at school, who had previously been rated soon after starting school. It examined whether teachers' ratings displayed predictive stability over that period, and whether significant differences evident at age 5 in SES and gender were still operative at age 7. SAMPLE: Two samples, of low income (N = 85) and middle income (N = 63) children, were rated following school entry (mean age 5 years 3 months) and rated again after two years at school. METHOD: The children were rated at both points by their regular classroom teachers using the Learning Behaviours Scale (Stott et al., 1998) with subscales of Distractible, Apprehensive and Uncooperative, together with ratings of academic achievement and their personal perception of each child. RESULTS: SES was found to be a very limited predictor for the learning behaviour subscale ratings and for teachers' personal perceptions at both ages 5 and 7. SES did significantly predict expected Academic Achievement at age 5, but this effect disappeared completely by age 7. Conversely, within the two defined groups, Low Income boys were found to display significantly poorer learning behaviours at age 5, especially in terms of distractible behaviour, compared with Middle Income boys and with girls generally. This pattern was maintained over the next two years of their schooling. The effect of SES was thus demonstrated more powerfully in between-group differences than by means of regression. The findings emphasised the persistence of teachers' initial negative impressions about distractible 'hard to manage' boys from low SES families. CONCLUSION: The outcomes of this study suggest that low SES boys commenced school significantly disadvantaged by a pattern of perceived distractible behaviour in particular, and that this perception continued to operate over the next two years of their schooling. Such a pattern implied that these boys were perceived by their teachers, from early in their school careers, as being demanding and difficult to teach. Once teachers gave certain boys a label it appeared to 'stick'. SES per se was thus not the main risk factor. It was primarily the effect of boys' greater activity level, distractibility, and initial inability to 'settle' to classroom routines, which seemed to be particularly associated with certain lower SES child-rearing practices. These behaviours had a serious negative impact on the children's teachers and the way they responded to them.

Rapamycin blocks IL-2-driven T cell cycle progression while preserving T cell survival.

Effective cellular immune responses require increases in antigen-specific T lymphocytes; IL-2 drives antigen-stimulated T cell proliferation and is largely responsible for the increases observed. We used microarrays containing approximately 9000 mouse cDNAs to study IL-2-induced gene expression. IL-2 induces the expression of genes that regulate cell cycle progression, control cell survival, and increase synthetic and metabolic processes during proliferation. IL-2 also suppresses expression of genes that block cell cycle progression and promote cell death. Rapamycin inhibits IL-2-driven proliferation by downregulating the expression of genes required for key processes required for cell cycle progression. Rapamycin also preserves cell survival by keeping intact the IL-2-induced cell survival programs. These complex multifaceted programs of gene expression permit a dynamic regulation of cellular proliferation and cellular survival.

The use of counterflow centrifugation to enrich gonadotropes and somatotropes.

Counterflow centrifugation produces populations of gonadotropes or growth hormone (GH) cells enriched to 90% in a Beckman elutriator. The pituitary populations are first separated by size into three fractions applying different flow rates, stimulated with either gonadotropin-releasing hormone (GnRH) to enlarge the gonadotropes or growth hormone-releasing hormone (GHRH) to enlarge the somatotropes for 3 hr. The fractions are re-eluted, first at the original flow rates and then at higher flow rates to separate enlarged gonadotropes or somatotropes. Most other cell types are reduced to less than 5%. However, co-storage of GH and gonadotropin antigens is seen in either population. Enriched gonadotropes or somatotropes can be used in studies of proliferation, autocrine or paracrine regulation, or ion channel functions.(J Histochem Cytochem 49:663-664, 2001)

Differential expression of estradiol receptors alpha and beta by gonadotropes during the estrous cycle.

This study focused on expression of estradiol receptors (ER) during the estrous cycle. Labeling for ERalpha or beta antigens and luteinizing hormone (LH) or follicle-stimulating hormone (FSH) beta-subunits was done on freshly dispersed pituitary cells. The lowest expression of ERalpha and beta was seen in estrus (23% and 12%, respectively). Expression increased to 42-54% of pituitary cells by diestrus. In males, cells with ERalpha or beta were 37% or 20% of the population, respectively. ERalpha or beta and gonadotropin antigens were in 6-9% of pituitary cells from male rats. Early in the cycle (estrus and metestrus), less than 5% of pituitary cells expressed ERalpha or beta with gonadotropins. These values doubled to reach a peak of 10% during proestrus (just before ovulation). These data show that a rise in expression of both ERalpha and ERbeta is a part of preovulatory differentiation of pituitary gonadotropes.(J Histochem Cytochem 49:665-666, 2001)

Sites of epidermal growth factor synthesis and action in the pituitary: paracrine and autocrine interactions.

1. Epidermal growth factor (EGF) is produced by growth hormone (GH) cells and gonadotropes in normal pituitary cell populations. The studies were initiated to determine whether EGF is a paracrine or autocrine regulator of gonadotrope function. 2. The first group of studies tested for the presence of EGF receptors in gonadotropes from cycling female rats by immunolabelling. Expression varied with the stage of the cycle. At the highest point (metoestrus), only a few EGF target cells are gonadotropes, identified by their content of luteinizing hormone (LH)-beta mRNA. Expression by gonadotropes then increased to reach a peak of 50% of cells during pro-oestrus. 3. Studies investigating the regulation of expression of EGF receptor (R) showed that all culture conditions (in media with or without serum) and EGF itself both stimulated expression of the receptor by gonadotropes in populations from oestrus or metoestrus rats. Gonadotropin-releasing hormone (GnRH) also stimulated EGFR expression in follicle-stimulating hormone (FSH) gonadotropes from oestrus animals. Additional tests of expression of immediate early genes (c-fos) showed that, after 15 min, EGF stimulated expression in cells with FSH antigens. 4. Epidermal growth factor also stimulated gonadotrope proliferation, as detected by the MTT cell growth/cell death assays and bromodeoxyuridine uptake by gonadotropes during the S phase (DNA synthesis) of the cell cycle. 5. Epidermal growth factor and GnRH both stimulated a significant increase in the percentage of mitotic gonadotropes. Epidermal growth factor may be an autocrine or a paracrine growth factor to maintain and develop the gonadotrope population and EGF may also be involved in early differentiation events that prepare cells to support the LH surge.

Epidermal growth factor and gonadotropin-releasing hormone stimulate proliferation of enriched population of gonadotropes.

Recent studies of epidermal growth factor (EGF) receptors on gonadotropes show that they appear early in the estrous cycle on immature gonadotropes, most of which could be identified by LH messenger RNA only. As diestrous gonadotropes translate the messenger RNAs, the percentages of LH and FSH cells with EGF receptors increase to reach a peak during proestrus. To learn more about the function of EGF in gonadotrope regulation, parallel studies of its mitogenic potential were conducted. To test this in a cell growth assay, we initially developed a protocol for enrichment of gonadotropes by counterflow centrifugation (elutriation). Analysis of immunolabeled cells in the enriched fraction showed that the population contained 90-95% cells with LH and/or FSH antigens. Less than 4% have TSH or PRL antigens, and less than 7% have ACTH antigens. About 15% of the enriched population expressed GH antigens in male rats and nearly 30% of the population express GH in females. This agrees with the known hormone storage overlap between these cells, especially in proestrous female rats. The MTT cell growth/cell death assay was then used to test the mitogenic potential of EGF, GnRH, and activin. This assay showed a linear relationship between plated cell numbers and optical density of the media after the MTT reaction was run. The enriched gonadotropes were plated in 96-microwell trays and grown for 3-4 days in the presence of defined media alone (no serum), or defined media containing 0.5-10 ng/ml EGF, 0.5-1 nM GnRH, 60 ng/ml activin or two of these factors. In all of the 12 experiments, each of the factors stimulated a 3- to 10-fold increase in optical density values, depending on the dose of the stimulating factor. The effects of any two factors were not additive. Because the MTT assays do not discriminate between mitogenic effects and enhanced cell survival, a second group of tests was run with mixed cultures of pituitary cells from diestrous female rats. These cells were cultured in the same combinations of EGF with and without GnRH for 3 h. During the last hour of culture, they were exposed to bromodeoxyuridine (BrDU) to identify cells that were synthesizing DNA. Cells in the S phase were thereby detected with dual immunocytochemical labeling for nuclear BrDU and gonadotropins. The analysis of dual labeled cells showed a 3-fold increase in percentage of LH or FSH cells with BrDU labeled nuclei following EGF or GnRH stimulation. The effects of the two growth factors were not additive. Collectively, these data confirm previous studies showing mitogenic functions for activin and now add EGF and GnRH as mitogens for gonadotropes.

Mental health care utilization and expenditures by children in foster care.

OBJECTIVE: To determine the percentage of children with mental health diagnoses and utilization and expenditures of mental health services among children in foster care compared with other children receiving Medicaid, including those with disabilities. DESIGN: Analysis of Medicaid claim and eligibility records in southwestern Pennsylvania for fiscal year 1995. POPULATION: A total of 39,500 children between ages 5 and 17 years continuously eligible for Medicaid in southwestern Pennsylvania were included in the analysis. MAIN OUTCOME MEASURES: Percentage of children with mental health diagnoses and mental and general health care utilization and expenditures classified by participation in foster care and Medicaid eligibility. RESULTS: Children in foster care were 3 to 10 times more likely to receive a mental health diagnosis, had 6.5 times more mental health claims, were 7.5 times more likely to be hospitalized for a mental health condition, and had mental health expenditures that were 11.5 times greater ($2082 vs $181) than children in the Aid to Families With Dependent Children (AFDC) program. Overall, utilization rates, expenditures, and prevalence of psychiatric conditions for children in foster care were comparable with those of children with disabilities. CONCLUSIONS: Children in foster care are significantly more likely to suffer from mental health conditions and use more mental health and general health services than AFDC children. Service use and expenditures are comparable with those of disabled children, suggesting that reimbursement rates and care management for children in foster care need to be reexamined.

Uninsured children with psychosocial problems: primary care management.

OBJECTIVE: Nearly 14% of children in the United States are uninsured. We compared the prevalence of psychosocial problems and mental health services received by insured and uninsured children in primary care practices. METHODS: The Child Behavior Study was a cohort study conducted by Pediatric Research in Office Settings and the Ambulatory Sentinel Practice Network. Four hundred one primary care clinicians enrolled an average sample of 55 consecutive children (4-15 years old) per clinician. RESULTS: Of the 13 401 visits to clinicians with 3 or more uninsured patients, 12 518 were by insured children (93.4%) and 883 were by uninsured children (6. 6%). A higher percentage of adolescents, Hispanic children, those with unmarried parents, and those with less educated parents were uninsured. According to clinicians, uninsured children and insured children had similar rates of psychosocial problems (19%) and severe psychosocial problems (2%). For children with a clinician-identified psychosocial problem, we found no differences in clinician-reported counseling, medication use, or referral to mental health professionals. CONCLUSIONS: Among children served in primary care practices, uninsured children have similar prevalence of clinician-identified psychosocial and mental health problems compared with insured children. Within their practices, clinicians managed uninsured children much the same way as insured children. psychosocial problems, uninsured children, pediatrics, family medicine, primary care.

Primary care treatment of pediatric psychosocial problems: A study from pediatric research in office settings and ambulatory sentinel practice network.

OBJECTIVE: Psychosocial problems cause much of the morbidity among children, and their frequency of presentation in primary care is growing. How is primary care treatment of children's psychosocial problems affected by child symptoms, physician training, practice structure, insurance, physician/patient relationship, and family demographics? DESIGN: Questionnaire study of treatment of psychosocial problems during office visits by children. SETTINGS: At total of 401 primary care offices from 44 US states, Puerto Rico, and Canada. PATIENTS: From 21 150 children seen in office visits, we selected children with an identified psychosocial problem but who were not already receiving specialty mental health services (n = 2618 children). OUTCOME MEASURES: Clinicians' decisions to counsel families, to refer children to mental health specialists, or to prescribe medication. RESULTS: The treatment choices of primary care clinicians (PCCs) were generally independent of patients' demographics or insurance status. Clinicians' training, beliefs about mental health, and practice structure had no effect on treatment choices. However, clinicians seeing their own patients were more likely to prescribe medications for attention problems. The clinician's perception about whether the parent agreed with the treatment choice was important for every treatment modality. Counseling and referral were more common and medication was less common when a problem was newly recognized at the visit. CONCLUSIONS: Structural factors such as practice type, insurance coverage, and physician training were less important for treatment than were process factors, such as whether the visit was a psychosocial problem visit, whether the problem was newly or previously recognized, and whether the family and clinician were familiar with each other and in accord about treatment.

Increasing identification of psychosocial problems: 1979-1996.

OBJECTIVE: To examine the changes in identification of pediatric psychosocial problems from 1979 to 1996. RESEARCH DESIGN: Comparison of clinician-identified psychosocial problems and related risk factors among large primary care pediatric cohorts from 1979 (Monroe County Study) and 1996 (Child Behavior Study). Data were collected from clinician visit questionnaires developed originally for the 1979 study. SETTING: Private practice offices of 425 community-based pediatricians and family practitioners across both studies. PATIENTS: We enrolled all children from 4 to 15 years of age who presented for nonemergent services in primary care offices. The 1979 study included 9612 children seen by 30 clinicians and the 1996 study included 21 065 children seen by 395 clinicians. SELECTION PROCEDURE: Each clinician enrolled consecutive eligible patients for both studies. MEASUREMENTS AND RESULTS: From 1979 to 1996, clinician-identified psychosocial problems increased from 6.8% to 18. 7% of all pediatric visits among 4- to 15-year-olds. We found increases in all categories of psychosocial problems, except for mental retardation. Attentional problems showed the greatest absolute increase (1.4%-9.2%) and emotional problems showed the greatest relative increase (.2%-3.6%). The use of psychotropic medications, counseling, and referral also increased substantially. In particular, the percentage of children with Attention deficit/hyperactivity problems receiving medications increased from 32% to 78%. These increases in psychosocial problems were associated with increases in the proportions of single-parent families and Medicaid enrollment from 1979 to 1996. Changes in clinician characteristics did not appear to be the source of increases in clinician diagnoses of psychosocial problems. CONCLUSIONS: Substantial increases in the identification of psychosocial problems in primary care paralleled demographic changes in children presenting to primary care offices and in the larger population.

Growth hormone cells as co-gonadotropes: partners in the regulation of the reproductive system.

Through unique receptors, growth hormone (GH) stimulates ovarian follicles and Leydig cells, working alone or synergistically with luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The source of GH might include a unique cell type that expresses mRNA encoding gonadotropin and GH and the antigens themselves, together with gonadotropin-releasing hormone (GnRH) and GH-releasing hormone (GHRH) receptors. This multifunctional cell might provide a cocktail of hormones needed to effect optimal gonadotropic activity.