Lack of association between gestational age adjusted TSH percentiles and neurodevelopmental outcomes among preterm infants.

BACKGROUND: Limited evidence exists on whether subclinical hypothyroidism suggested by mildly elevated TSH levels affect neurodevelopment and growth in preterm infants. The objective of this study was to determine the association between gestational age adjusted TSH percentiles and neurodevelopmental outcomes among preterm infants. METHODS: Univariate linear regression analysis was conducted to determine, in infants born less than thirty-two weeks gestational age, the correlation between the TSH percentile on the last newborn screen and neurodevelopmental assessment scores and growth outcomes at eighteen to twenty-two months of corrected age. RESULTS: Seventy-four patients were enrolled in the study with a mean gestational age of 28.8 weeks. There was no correlation between the last TSH percentile value and Bayley-III cognitive composite score or other neurodevelopmental or growth outcomes. CONCLUSION: In a cohort of preterm infants, higher TSH percentiles suggesting potential subclinical hypothyroidism did not predict any adverse effect on neurodevelopmental or growth outcomes.

Immune responses to human papilloma viruses.

HPV infection in the genital tract is common in young sexually active individuals, the majority of whom clear the infection without overt clinical disease. However most of those who develop benign lesions eventually mount an effective cell mediated immune response and the lesions regress. Regression of ano-genital warts is accompanied histologically by a CD4+ T cell dominated Th1 response; animal models support this and provide evidence that the response is modulated by CD4+ T cell dependent mechanisms. Failure to develop effective CMI to clear or control infection results in persistent infection and, in the case of the oncogenic HPVs, an increased probability of progression to CIN3 and invasive carcinoma. The central importance of the CD4+ T cell population in the control of HPV infection is shown by the increased prevalence of HPV infections and HGSIL in individuals immunosuppressed as a consequence of HIV infection. The prolonged duration of infection associated with HPV seems to be associated with effective evasion of innate immunity as reflected in the absence of inflammation during virus replication, assembly and release, and down regulation of interferon secretion and response thus delaying the activation of adaptive immunity. Serum neutralising antibody to the major capsid protein L1 usually develops after the induction of successful cell mediated immunity and these antibody and cell mediated responses are protective against subsequent viral challenge in natural infections in animals. Prophylactic vaccines consisting of HPV L1 VLPs generate high anti L1 serum neutralizing antibody concentrations and in clinical trials have shown greater than 95 per cent efficacy against both benign and neoplastic genital HPV associated disease. These vaccines are delivered intramuscularly and therefore circumvent the immune evasion strategies of the virus.

Integration of high-risk human papillomavirus DNA is linked to the down-regulation of class I human leukocyte antigens by steroid hormones in cervical tumor cells.

A crucial event in the malignant progression of cervical intraepithelial neoplasia appears to be the up-regulation of high-risk human papillomavirus (HPV) early gene expression. Steroid hormones have been linked to the progression from premalignant to neoplastic status in HPV positive lesions. This report demonstrates that at physiological levels, the glucocorticoid hormone hydrocortisone consistently down-regulates class I human leukocyte antigen (HLA) surface expression in HPV-positive cervical tumor cells but can up-regulate expression in HPV-negative epithelial tumor lines. Suppression of HLA expression was also seen with progesterone, another steroid hormone. The hydrocortisone-mediated modulation of HLA expression is dependent on integration and transcription of the HPV genome and can be blocked by Ru38486, an antagonist of both glucocorticoid and progesterone receptors, indicating the role of these receptors in mediating HLA suppression. The data suggest that HPV integration events in cervical epithelia correlate with hormone-dependent HLA suppression, possibly contributing to the avoidance of tumor recognition by cytotoxic T cells. These studies imply that clinical use of steroids may be contraindicated in HPV-positive individuals who have early premalignant cervical disease or neoplasia but provide evidence that the antiprogestin Ru38486 may be useful in the management of early stage cervical disease.

In vivo and in vitro effects of v-fos and EJ-Ha-ras oncogene expression in murine epidermal keratinocytes.

Primary neonatal Balb/c keratinocyte (NEK) cultures grown, using 3T3 feeder cell support in high calcium, serum supplemented medium, were transfected with EJ-Ha-ras or v-fos DNA sequences and pSV2 neo. Several neo resistant clones were isolated and several established cell lines expressing the transfected gene products derived. Two of these lines, Ras 8 and Fos 1, have been examined in detail with respect to their self renewal capacity and differentiation potential in vitro and in vivo. In vitro, both lines (when compared to normal NEK) have an extended probably immortal phenotype, enhanced colony forming efficiency (a measure of in vitro self renewal capacity) and a reduction in growth factor and serum dependence. When grafted onto syngeneic recipients neither cell line is tumourigenic. Instead a histologically abnormal epithelium with no stratum corneum and with features specific to the oncogene expressed is formed. The extent of the histological atypia correlates with the in vitro alterations in cytoskeletal peptides as revealed by 2D PAGE. However despite the gross histological abnormality there is no alteration in the in vivo self renewal capacity (measured as the number of grafted cells required for epidermal reformation) between normal NEK and the Ras 8 or Fos 1 lines; in each case a minimum of 10(5) cells/1.14 cm2 is required before a full thickness epithelium forms.

Amplification of chromosome 5p correlates with increased expression of Skp2 in HPV-immortalized keratinocytes.

The oncogenic HPVs immortalize primary genital keratinocytes in vitro and there is evidence that such lines represent suitable models to examine HPV-induced carcinogenesis. Early in vivo studies and more recent CGH analyses have revealed amplification of chromosome 5p in advanced stage carcinoma of the uterine cervix (CaCx). In the present study, a panel of established CaCx-derived cell lines were analysed by M-FISH to identify recurrent karyotypic abnormalities. Amplification of 5p was observed in 11 of 13 CaCx cell lines harbouring HR (high-risk) HPV. The region of 5p undergoing amplification was confirmed using human band-specific paints. The F-box protein Skp2 is present at 5p13 and its protein is present at increased levels in many cancers of an advanced stage. The HPV16-harbouring cell line W12 shows progressive morphological abnormality with in vitro passage, culminating in an invasive phenotype. The expression of Skp2 at different stages of this progression was investigated utilizing Western blot and TaqMan quantitative PCR. At medium to late passage, gain of 5p as an isochromosome was observed. Increased expression of Skp2 and a reduction in the expression of its target p27 correlated with increasing passage in this line.

Association of CDKN2A/p16INK4A with human head and neck keratinocyte replicative senescence: relationship of dysfunction to immortality and neoplasia.

We have previously suggested that a gene mapping to chromosome 9p21 could contribute to replicative senescence and suppress cullular immortality in squamous neoplasia. Two candidate genes, the cyclin D1/cyclindependent kinase inhibitors CDKN2A/p16INK4A (p16) and CDKN2B/p15INK4B (p15) have now been identified in this region and we show here that p16 is upregulated when normal human keratinocytes undergo replicative senescence but not when they undergo differentiation. Furthermore, all of 19 immortal neoplastic keratinocyte head and neck lines, including nine showing loss of heterozygosity (LOH) at 9p21, showed undetectable p16 expression, whereas five of six senscent neoplastic cultures showed normal levels of expression. The retinoblastoma protein (pRb) appeared functional in all the cell lines and cultures examined. The mechanism of p16 inactivation appeared to be transcriptional silencing in 10 of 18 lines and homozygous deletions in the rest. Treatment of two of the immortal cell lines which had transcriptionally silent wild type p16 genes with 5aza-2deoxycytidine resulted in the re-expression of p16, thus implicating DNA methylation as one mechanism of transcriptional silencing in the immortal SCC-HN lines. We observed no cases of p16 point mutation. In contrast, the p15 gene was rarely transcriptionally silent and was not deleted in any of the cell lines which showed p16 deletions. Our results show that p16 dysfunction correlates strongly with keratinocyte immortalisation but less strongly with the stage of tumour progression. P16 dysfunction was not related to the neoplastic state or the length of time spent in vitro. The results also suggest that p16 but not p15 is involved in the keratinocyte replicative senescence programme. However, two neoplastic cell cultures which lacked p16 expression were still mortal, suggesting that the loss of p16 is a necessary but insufficient condition for human keratinocyte immortality.

Immunoelectron microscopical localization of human papillomavirus type 16 L1 and E4 proteins in cervical keratinocytes cultured in vivo.

The human papillomavirus (HPV) causes warts, but is also associated with the development of squamous cell dysplasia and carcinoma. The virus is host and tissue specific and the numerous HPV types show predilection for different body sites. Experimental production of HPV 16 particles is at present only possible using in vivo culture of keratinocytes containing episomal viral DNA. Using immunoelectron microscopy, we have investigated the localization of HPV 16 E4 and L1 proteins in a keratinized epithelium formed by grafting HPV 16-containing cervical keratinocytes onto the athymic mouse. New viral progeny are produced in this system, as confirmed by labeling of intranuclear particles with a mouse monoclonal antibody against the HPV 16 major capsid (L1) protein. The role of the E4 protein is not yet clear, although it is believed to be important for the later stages of the virus life cycle. Here we confirm its cytoplasmic localization in the cells of the spinous and granular layers and demonstrate co-localization with keratin tonofilaments.

The influence of MHC-compatible and MHC-incompatible antigen-presenting cells on the survival of MHC-compatible cultured murine keratinocyte allografts.

Our group has shown previously that APC-depleted cultured epidermal keratinocytes show prolonged survival when grafted onto normal MHC-incompatible adult mice. We show here that in vitro culture also improves significantly the survival of MHC-compatible keratinocyte allografts, although these nonrejected grafts are repopulated by host cells identified by their dendritic morphology and phenotype (class II+, leukocyte-common antigen+) as APCs. Reconstitution of cultured grafts, immediately prior to transplantation, with MHC-compatible dendritic cells of either donor or recipient origin, results in graft rejection--thus demonstrating that cultured cells can be rejected by the recipient animal--and suggests that a paucity of APCs in the immediate postgrafting period is responsible for the privilege afforded these grafts.

Prolonged survival of cultured keratinocyte allografts in the nonimmunosuppressed mouse.

The effect of in vitro culture on the survival of allografts of epidermal keratinocytes has been examined using a mouse model. Female BALB/c tail epidermal cells were cultured from single cell suspensions to form confluent sheets that were grafted onto male CBA recipients using a transplantation technique that ensured separation of donor graft from host skin. Animals were killed at defined intervals, and the status of the grafts determined histologically. Full thickness skin allografts rejected at 13-15 days. Allografts of epidermis (obtained by enzymatic cleavage at the dermoepidermal junction) rejected at 19-20 days. Cultured keratinocyte allografts were not rejected at least within 70 days and had a histological appearance identical to syngeneic controls. The expression of MHC class I and class II determinants and the leukocyte common (Ly5) surface marker on the donor cells before and after culture were examined using indirect immunofluorescence and monoclonal antibodies. These and other cytochemical studies showed that freshly dissociated tail epidermal cells contained 0.3% of cells that expressed membrane-bound ATP-ase activity, Ia antigens, and the Ly5 surface antigen. These are the Langerhans' cells of the epidermis. In culture, these cells decrease so that by day 8 of culture, no such cells can be detected. At confluence, there are no Ia positive cells, but all cells express MHC class I antigens and stain with an antikeratin antibody. The loss of Ia expression on culture correlates with a decreased stimulation of the class II H-2d-restricted T cell clone D7.1 by cultured keratinocytes compared with freshly dispersed epidermal cells. Furthermore, cultured keratinocytes fail to prime allogeneic mice as determined by the survival of whole thickness skin grafts, whereas freshly dispersed cells induce an accelerated rejection. The results suggest that the survival of cultured keratinocyte allografts is due to the elimination of cells expressing Ia antigens and supports the passenger leukocyte theory of graft rejection.

Expression of the myelomonocytic antigens CD36 and L1 by keratinocytes in squamous intraepithelial lesions of the cervix.

The keratinocytes in squamous intraepithelial lesions (SILs) of the cervix show altered expression of a number of molecules involved both in the control of growth and differentiation and in cell surface interactions, particularly with components of the immune system. We have used tissue biopsies and in vitro model systems to investigate the expression in SILs of the molecules CD36 and L1, which are predominantly expressed by myelomonocytic cells but which also have functional roles in keratinocyte biology. Whereas the L1 protein (defined by the monoclonal antibody Mac387) was expressed by suprabasal and superficial cells in 12 of 12 cases of normal cervix (NCx) and in 14 of 14 cases of low-grade SILs (LG-SILs), in two of 16 cases of high-grade SILs (HG-SILs) it was entirely absent and in the remainder it was restricted to the most superficial layers. When an arbitrary grading scale was applied, L1 expression in HG-SILs proved to be significantly lower than in LG-SILs (P < .01) or in cases of NCx (P < .01). CD36 was expressed by superficial cells in four of 12 cases of NCx, in six of 14 LG-SILs, and none of 16 cases of HG-SILs (when graded, LG-SILs v HG-SILs = P < .05). The mechanisms underlying the expression of both molecules were investigated by growth in organotypic tissue culture of normal ectocervical epithelium and the cervical keratinocyte cell lines W12 (a model for LG-SILs) and CaSki and SiHa (models for HG-SILs). L1 was diffusely expressed by NCx cells and the W12 cell line, although its expression in the CaSki and SiHa cell lines was much more irregular and restricted. CD36 was occasionally present on the surface of superficial NCx and W12 cells, but was absent from CaSki and SiHa cells. Neither molecule could be induced by treatment of the cells with interferon-gamma. These data suggest that the expression of CD36 and L1 by cervical keratinocytes is related to their differentiation status rather than representing an effect of exogenous factors, such as those released by the immune cell infiltrate associated with SILs. CD36 may function as an immunoregulatory molecule on cervical keratinocytes in SILs, while L1 is more likely to be involved in the intracellular regulation of cell proliferation and maturation.

Enhancement of the innate and cellular immune response in patients with genital warts treated with topical imiquimod cream 5%.

The mechanism of action of imiquimod 5% cream applied topically to patients with genital warts was evaluated in a double-blind, placebo-controlled study. Imiquimod (16 patients) or placebo (three patients) was applied three times per week for up to 16 weeks. All imiquimod-treated patients had a > or =75% reduction in total wart area while only one of three placebo-treated patients had a similar reduction. Wart biopsies were taken at prestudy, week 6, and end of treatment. Polymerase chain reaction (PCR) for human papillomavirus (HPV) DNA and reverse transcriptase (RT)-PCR for messenger (m)RNAs were used to identify cytokines, cellular markers, viral gene products, and cell cycle markers in these biopsies. Treatment with imiquimod, an immune response modifier, stimulated significant increases in mRNA for interferon (IFN)-alpha, IFN-gamma and 2',5' oligoadenylate synthetase (2',5'-AS) as well as a tendency towards increases in tumor necrosis factor (TNF)-alpha and interleukin-12 p40. Significant increases in mRNA for CD4 and a trend toward increases in CD8 were also observed in imiquimod-treated patients, suggesting activation of a cell mediated immune response. Imiquimod administration was also associated with a significant decrease in viral load as measured by HPV DNA and L1 mRNA. The effects on HPV markers were accompanied by an apparent decrease in mRNA expression for markers of cell proliferation and an increase in mRNA for markers of keratinocyte differentiation and tumor suppressors.

Immunological events in regressing genital warts.

Little is known of the in vivo role of the immune system in controlling human papillomavirus infection in the genital tract. The authors have studied 125 closely monitored patients with genital warts. Of these 125 patients, wart regression was seen in 28 patients. This study provides evidence that clearance of human papillomavirus from the genital tract is characterized by an active cell-mediated immune response. Regressing warts (n = 14) contained significantly more T lymphocytes (P < .05, Wilcoxon rank sum test) and macrophages (P < .01) than did nonregressing controls (n = 14). CD4-positive lymphocytes predominated in regression, both within the wart stroma and the surface epithelium, where there was a significant change in the ratio of CD4+ to CD8+ cells (P < .01). Lymphocytes in regression also showed greater expression of activation markers, and the majority were of the "antigen-experienced" phenotype. There was no difference in Langerhans cell numbers, although there was significant induction of the immune accessory molecules HLA-DR and ICAM1 (P < .05) on keratinocytes, and E-selectin and VCAM1 (P < .05) on endothelial cells in regressing warts. The changes in regression are consistent with a delayed-type hypersensitivity reaction to foreign antigen, and the ability to induce and mount such a response may be a critical determinant of effective natural immunity to the genital HPVs. Specific targeting of delayed-type hypersensitivity responsiveness may increase the efficacy of strategies for immuno-intervention against HPV infection in the genital tract.

Altered expression of desmosomal components in high-grade squamous intraepithelial lesions of the cervix.

We have used immunohistochemistry to test the hypothesis that components of the desmosome are disrupted during neoplastic progression of squamous epithelial cells in the uterine cervix. Sections of normal cervix and squamous intraepithelial lesions (SILs) were immunostained for desmosomal proteins and glycoproteins, and results were assessed using a semi-quantitative grading system. No difference between normal cervix and low-grade SIL (LSIL) was found. A significant reduction in expression of desmogleins was seen between high-grade SIL (HSIL) and LSIL (P<0.01) and normal cervix (P<0.001). Desmocollin expression was not reduced significantly, although scores showed significantly greater variation in HSIL compared with LSIL (P<0.05) and normal cervix (P<0.05). There was no significant difference in desmoplakin expression among the three groups. The results suggest that there may be sequential disruption of desmosomal function during neoplastic progression of cervical squamous intraepithelial cells, with downregulation of desmogleins during the progression from LSIL to HSIL and loss of desmocollin expression occurring in some cases of established HSIL.

Anxiety disorders.

Research has only recently begun to address the nature and treatment of anxiety in later life. Prevalence rates suggest that anxiety disorders occur more than twice as frequently as depression among older adults, with the highest rates reported for generalized anxiety disorder and phobias. However, relatively little is known about the psychopathology and treatment of these and other anxiety disorders. In this review, recent clinical advances in this area are summarized. Particular attention is given to the prevalence and nature of late-life anxiety and its overlap with related conditions, psychometric properties of assessment tools for evaluating anxiety in older adults, and treatment outcome literature in both pharmacological and psychosocial domains. Directions for future research are provided throughout and summarized in a final section with the goal of stimulating additional empirical work in the area.

Comparison of cognitive therapy and relaxation training for panic disorder.

Current approaches to the treatment of panic disorder (PD) include a treatment package consisting of relaxation training (RT), cognitive therapy (CT), and exposure-based components. In an examination of the separate effects of RT and CT without formally taught exposure of any form, 64 PD patients were assigned randomly to one of these treatment protocols or to a minimal-contact control (MCC) condition. Both RT and CT were superior to the MCC condition on a variety of measures pertaining to panic, global psychological functioning, agoraphobic fear, and other associated fears. A significantly greater percentage of patients were classified as treatment responders (based on a composite index) after CT (82%) and RT (68%), compared with the control group (36%). On measures of agoraphobic fear, CT patients fared slightly better than RT patients. Some support was demonstrated for specific cognitive changes after CT, although treatment specificity was not strongly supported overall. These results are discussed in light of current theories of PD and the presumed importance of exposure in its treatment.

Social phobia: Axis I and II correlates.

The presence of Axis I and Axis II disorders in 71 social phobic patients was examined. Generalized anxiety disorder was the most common secondary Axis I disorder, followed by simple phobia. Avoidant personality disorder and obsessive-compulsive personality disorder were the most common Axis II diagnoses, and 88% of the sample exhibited features of these 2 personality styles. Subjects with additional Axis I diagnoses were more anxious and depressed than those with no additional Axis I disorder. Social phobics with additional Axis II disorders were more depressed but not more anxious than those with no Axis II diagnosis. Furthermore, those with an additional Axis I disorder had higher scores on measures of neuroticism, interpersonal sensitivity, and agoraphobia. The prevalence and impact of additional Axis I and II disorders on the etiology, maintenance, and treatment outcome for persons with social phobia are discussed.

Alprazolam-reinforced medication use in outpatients with anxiety.

The reinforcing effects of alprazolam were investigated in 14 patients who had generalized anxiety or panic disorder, but were not current users/abusers of other psychoactive substances. Using a double-blind outpatient choice procedure, color-coded alprazolam (0.5 mg) and placebo capsules were provided to patients for use 'as needed' in the treatment of anxiety symptoms. Comparisons of alprazolam and placebo during a 2 week sampling period in which placebo and alprazolam were available sequentially revealed no significant differences on measures of medication usage or anxiety levels, although alprazolam did increase subjective ratings of drug effects side effects. During a 4 week choice period, alprazolam was strongly preferred over placebo in 11 out of 14 patients indicating that alprazolam functioned as a reinforcer. Medication usage ranged from zero to 4.0 mg alprazolam in a day. Variations in daily medication-use were positively correlated with anxiety level fluctuations for a majority of patients. For a majority of patients, the results indicate that alprazolam functioned as a reinforcer without accompanying signs of abuse or addiction.

Human papillomavirus and cervical carcinogenesis.

Epidemiological studies show that infection with a subset of genital human papillomavirus (HPV) infections is the major risk factor for the subsequent development of cervical cancer. Experimental studies show that that the E6 and E7 genes of these high risk HPVs are oncogenes that deregulate key cell cycle controls. In the normal infectious cycle high level expression of these genes is confined to non-dividing differentiated cells: HPV oncogenesis requires deregulation of viral and cellular genes permitting inappropriate expression of E6 and E7. These are rare events but viral persistence and chronic exposure to steroid hormones increase the probability of this deregulation.

The immunology of animal papillomaviruses.

Papillomaviruses are species- and tissue-specific double-stranded DNA viruses. These viruses cause epithelial tumours in many animals, including man. Typically, the benign warts undergo spontaneous, immune-mediated regression, most likely effected by T-cells (especially CD4, but also CD8 subsets), whereas humoral immunity can prevent new infections. Some papillomavirus infections fail to regress spontaneously and others progress to malignant epithelial tumours. Additionally, the impact of these lesions is greater in immunosuppressed individuals. Many therapies are ineffective, and there is much interest in the potential for immunological intervention in papillomavirus infections of man and animals. Vaccination can be achieved with 'live' virus, formalin-inactivated virus, synthetic virus-like particles, and DNA vaccination. There has been much recent progress in the development of such vaccines for papillomavirus infections in the rabbit, ox and dog. Success in these animal models suggests that similar approaches may prove useful for prophylactic or therapeutic vaccination against the important human papillomaviruses involved in the development of cutaneous and anogenital warts, laryngeal papillomatosis, and cervical cancer.

Affective correlates of trichotillomania.

Affective correlates of hair pulling were investigated in a sample of 44 participants diagnosed with trichotillomania (TM). Participants completed the Hair Pulling Survey on which they rated the intensity of ten different affective states across three different phases of hair pulling (before, during and after). Repeated measures analysis of variance was used to examine the change of emotional experience across the hair pulling cycle. Results indicated significant decreases in boredom, anxiety and tension, and significant increases in guilt relief, sadness and anger across time (p<0.005). The role of co-existent anxiety and mood disorders also was examined using repeated measures analysis of variance. Results of these analyses indicated that patients with and without co-existent disorders differed only on patterns of anger across time, and therefore do not support affective subtypes of TM patients based on co-existent diagnosis. Implications of these findings for conceptualization and treatment of TM are discussed.