Assessing cognitive decline in the aging brain: lessons from rodent and human studies.

As life expectancy continues to increase worldwide, age-related dysfunction will largely impact our societies in the future. Aging is well established to promote the deterioration of cognitive function and is the primary risk factor for the development of prevalent neurological disorders. Even in the absence of dementia, age-related cognitive decline impacts specific types of memories and brain structures in humans and animal models. Despite this, preclinical and clinical studies that investigate age-related changes in brain physiology often use largely different methods, which hinders the translational potential of findings. This review seeks to integrate what is known about age-related changes in the brain with analogue cognitive tests used in humans and rodent studies, ranging from "pen and paper" tests to virtual-reality-based paradigms. Finally, we draw parallels between the behavior paradigms used in research compared to the enrollment into clinical trials that aim to study age-related cognitive decline.

Molecular characterization of cbfß gene and identification of new transcription variants: implications for function.

The CBFß gene encodes a transcription factor that, in combination with CBFα (also called Runx, runt-related transcription factor) regulates expression of several target genes. CBFß interacts with all Runx family members, such as RUNX2, a regulator of bone-related gene transcription that contains a conserved DNA-binding domain. CBFß stimulates DNA binding of the Runt domain, and is essential for most of the known functions of RUNX2. A comparative analysis of the zebrafish cbfß gene and protein, and of its orthologous identified homologous proteins in different species indicates a highly conserved function. We cloned eleven zebrafish cbfß gene transcripts, one resulting in the known Cbfß protein (with 187 aa), and three additional variants resulting from skipping exon 5a (resulting in a protein with 174 aa) or exon 5b (resulting in a protein with 201 aa), both observed for the first time in zebrafish, and a completely novel isoform containing both exon 5a and 5b (resulting in a protein with 188 aa). Functional analysis of these isoforms provides insight into their role in regulating gene transcription. From the other variants two are premature termination Cbfß forms, while the others show in-frame exon-skipping causing changes in the Cbfß domain that may affect its function.

Characterization of polyhydroxyalkanoates accumulated by a moderately halophilic salt pan isolate Bacillus megaterium strain H16.

AIM: Characterization of polyhydroxyalkanoates (PHA) accumulated by halophilic bacteria isolated from solar salterns. METHODS AND RESULTS: Twenty-six halophilic isolates were obtained from solar salterns of Goa, India. They were screened for accumulation of PHA by Sudan black B, Nile blue A and Nile red stains. Strains H15, H16 and H26 were selected based on their intensity of Nile blue A/Nile red fluorescence. On the basis of phenotypic and genotypic characterization, the three isolates were identified as Bacillus megaterium. Growth kinetics and polymer accumulating capacity of strain H16 were studied in E2 mineral media with 2% glucose with/without NaCl. In the absence of NaCl, strain H16 accumulated PHA to 40·0% (w/w) of cell dry weight (CDW) at 42 h of growth, whereas in presence of 5% w/v NaCl, the culture showed longer lag phase of up to 24 h and accumulated a maximum PHA of 39% (w/w) CDW at 54 h of growth. The infrared spectra of both the polymers exhibited peaks at 1733·9 cm(-1) characteristic of C=O. Scans of (1) H nuclear magnetic resonance (NMR) showed a doublet at 2·5 ppm corresponding to methylene group (-CH(2)), the signal at 5·3 ppm corresponded to methine group (-CH-), and another signal at 1·3 ppm corresponded to the methyl group (-CH(3)). Scans of (13)C NMR showed prominent peaks at 20, 40, 67-68 and 170 ppm, indicating the polymer to be homopolymer of 3-hydroxybutyrates. The polymer is stable up to a temperature of 160°C. CONCLUSION: Three moderately halophilic isolates (strain H15, H16 and H26) capable of accumulating PHA were isolated from solar salterns of Ribandar Goa, India, and identified as B. megaterium based on phenotypic and genotypic characterization. Strain H16 accumulated polyhydroxybutyrate in the presence and absence of NaCl up to 40% of its CDW. SIGNIFICANCE AND IMPACT OF THE STUDY: This strain would be better suited for production of PHA at industrial level due to its tolerance to high concentration of NaCl.

Safety of screening with Human papillomavirus testing for cervical cancer at three-year intervals in a high-risk population: experience from the LAMS study.

OBJECTIVES: To assess whether human papillomavirus (HPV) testing is a safe enough approach to warrant extension of the screening intervals of baseline Papanicolaou (Pap)-/HPV- women in low-income settings. METHODS: Of the >1000 women prospectively followed up as part of the Latin American Screening (LAMS) Study in São Paulo, Campinas, Porto Alegre) and Buenos Aires, 470 women with both baseline cytology and Hybrid Capture 2 (HC2) results available were included in this analysis. These baseline Pap-negative and HC2- or HC2+ women were controlled at six-month intervals with colposcopy, HC2 and Pap to assess the cumulative risk of incident Pap smear abnormalities and their predictive factors. RESULTS: Of the 470 women, 324 (68.9%) were high-risk HPV (hrHPV) positive and 146 (31.1%) were negative. Having two or more lifetime sex partners (odds ratio [OR] = 2.63; 95% CI 1.70-3.51) and women using hormonal contraception (OR = 2.21; 95% CI 1.40-3.51) were at increased risk for baseline hrHPV infection. Baseline hrHPV+ women had a significantly increased risk of incident abnormal Pap smears during the follow-up. Survival curves deviate from each other starting at month 24 onwards, when hrHPV+ women start rapidly accumulating incident Pap smear abnormalities, including atypical squamous cells (ASC) or worse (log-rank; P < 0.001), low-grade squamous intraepithelial lesions (LSIL) or worse (P < 0.001) and high-grade squamous intraepithelial lesions (HSIL) (P = 0.03). Among the baseline hrHPV- women, the acquisition of incident hrHPV during the follow-up period significantly increased the risk of incident cytological abnormalities (hazard ratio = 3.5; 95% CI 1.1-11.7). CONCLUSION: These data implicate that HPV testing for hrHPV types might be a safe enough approach to warrant extension of the screening interval of hrHPV-/Pap-women even in low-resource settings. Although some women will inevitably contract hrHPV, the process to develop HSIL will be long enough to enable their detection at the next screening round (e.g. after three years).

Drug addiction is not an independent risk factor for oncogenic human papillomavirus infections or high-grade cervical intraepithelial neoplasia: case-control study nested within the Latin American Screening study cohort.

Drug abuse (addiction) has been listed among the risk factors for human papillomavirus (HPV) infections, but no case-control studies exist to rule out sexual behaviour and other potential confounders. The aim of this study is to evaluate the role of drug addiction as an independent predictor of HR-HPV infections and (cervical intraepithelial neoplasia) CIN2+ in an age-matched case-control (1:4) study nested within the prospective Latin American Screening (LAMS) study cohort. All 109 women in the LAMS cohort (n=12,114) reporting drug abuse/addiction were matched with four controls (n = 436) of non-abusers strictly by age. Conditional logistic regression analysis was used to estimate the co-variates of drug abuse, and the whole series (n=545) was analysed for predictors of HR-HPV and CIN2+ using univariate and multivariate regression models. Oncogenic HPV infections were significantly (P=0.019) more prevalent among abusers (37.7%) than in controls (21.9%), but there was no difference in high-grade squamous intraepithelial lesions (P=0.180) or CIN2+ lesions (P=0.201). In multivariate conditional logistic regression, number of lifetime sexual partners (P=0.0001), ever smokers (P=0.0001), non-use of OCs (P=0.013), ever having sexually transmitted diseases (STD) (P=0.041) and no previous Pap smear (P=0.027) were independent co-variates of drug addiction. Drug abuse was not an independent risk factor of high-risk (HR)-HPV infection, which was significantly predicted by (1) age below 30 years (P=0.045), (2) more than five lifetime sexual partners (P=0.046) and (3) being current smoker (P=0.0001). In multivariate model, only HR-HPV infection was an independent risk factor of CIN2+ (P=0.031), with adjusted OR=11.33 (95% CI 1.25-102.50). These data indicate that drug addiction is not an independent risk factor of either HR-HPV infections or CIN2+, but the increased prevalence of HR-HPV infections is explained by the high-risk sexual behaviour and smoking habits of these women.

Expression of p16 and cervical infection with high-risk human papillomavirus are not related to p53 activity in cervical intraepithelial neoplasia.

The objective of the study was to investigate the expression of p53 and p16(INK4a) in cervical intraepithelial neoplasia (CIN) and their relation with disease severity and high-risk human papillomavirus (HR-HPV) status. A series of 125 women with previous positive Pap smear were selected for this cross-sectional study. All patients underwent gynecological examination, including colposcopy. Specimens for Pap smears, Hybrid Capture 2 (HC2) test, and pathologic analysis were obtained. After evaluation of CIN grade, immunohistochemical detection of proteins p53 and p16(INK4a) was performed on paraffin-embedded sections. The extent of immunoexpression of both proteins was analyzed in relation to CIN grade and HR-HPV status. CIN was graded as 1 in 21, 2 in 17, and 3 in 87 specimens. p16(INK4a) positivity (at least 5% of epithelial cells stained) was found in 99 of 125 cases (79.2%) and was significantly higher in high-grade lesions as compared to low-grade CIN (P < 0.001). The expression of p53 did not differ across histologic strata. Protein expression neither of p16(INK4a) nor of p53 correlated with HR-HPV status. Expression of p16(INK4a) was not related with that of p53. Our study gives further support to previous findings of strong association of p16(INK4a) immunostaining with severity of epithelial atypia, but this protein may not be considered a predictor of HR-HPV status determined with HC2. By contrast, immunoexpression of p53 was related neither to CIN grade nor to HR-HPV status.

Human papillomavirus testing as an optional screening tool in low-resource settings of Latin America: experience from the Latin American Screening study.

Hybrid capture II (HC II) test for oncogenic human papillomaviruses (HPV) was carried out in a cohort of 4284 women at their first clinical visit. Overall prevalence of HPV was 17.1%, decreasing with age from 33.9% among women below 20 years to only 11.0% among those older than 41 years. HPV prevalence was significantly higher among current smokers (odds ratio [OR] = 1.31; 95% CI 1.1-1.6), in women with two or more lifetime sexual partners (OR = 1.9; 95% CI 1.6-2.4), and those women with two or more sexual partners during the past 12 months prior to examination (OR = 1.6; 95% CI 1.2-2.2). HPV detection increased in parallel with increasing cytologic abnormality, being highest in women with high-grade squamous intraepithelial lesion (P= 0.001). Specificity of the HPV test in detecting histologically confirmed cervical disease was 85% (95% CI 83.9-86.1). Sensitivity of the HPV test in detecting histologic abnormalities increased in parallel with disease severity, ranging from 51.5% for cervical intraepithelial neoplasia (CIN) 1 to 96.5% for CIN 3 and 100.0% for cancer, with respective decline of positive predictive value. These data suggest that HPV testing with HC II assay might be a viable screening tool among this population with relatively high prevalence of cervical disease.

Evaluation of visual inspection with acetic acid (VIA), Lugol's iodine (VILI), cervical cytology and HPV testing as cervical screening tools in Latin America. This report refers to partial results from the LAMS (Latin AMerican Screening) study.

OBJECTIVES: To assess the performance indicators of visual inspection with acetic acid (VIA) and visual inspection with Lugol's iodine (VILI) in four Latin American centres participating in the ongoing Latin AMerican Screening (LAMS) study, in settings with moderate incidence of cervical disease and with poorly to moderately well-organized cervical cancer screening. SETTING: Three Brazilian centres (São Paulo, Campinas and Porto Alegre) and one Argentine centre (Buenos Aires) recruited a total of 11,834 healthy women to undergo VIA, VILI, conventional Pap smear and Hybrid Capture II (HCII). METHODS: Women who had a positive result from any of these tests were subjected to colposcopy and biopsies (if necessary), and women with high-grade cervical intraepithelial neoplasia (CIN) were properly treated. To control for verification bias, 5% of women with normal tests were referred for colposcopy, as were 20% of HCII-negative women. RESULTS: Data on VIA (n=11,834), VILI (n=2994), conventional Pap smear (n=10,138) and HCII (n=4195) were available for test comparisons, calculating sensitivity, specificity, and positive and negative predictive values. Overall test positivity was 11.6% for VIA, 23.0% for VILI, 2.2% for Pap smear (LSIL threshold), 1.1% for Pap smear (HSIL threshold) and 17.1% for HCII. VIA was positive in 61.8% of the women with CIN 1, 57.0% of those with CIN 2, 35.0% of women with CIN 3 and in 21 of 28 (75%) of women with cancer. Approximately 10% of women with no detectable disease had an abnormal VIA. Regarding VILI, 83.3% of women diagnosed with CIN 1 and 62.5% of those with CIN 3 had an abnormal test. VILI failed to detect one of three cases of cancer. Both the sensitivity, specificity and positive predictive value of VIA and VILI in detecting CIN 2 or CIN 3 could be significantly improved depending on the combination with Pap smear or HCII (sensitivity up to 100.0% and specificity up to 99.8%). CONCLUSIONS: The LAMS study failed to reproduce the performance figures obtained with VIA and VILI (as stand-alone tests) in some other settings, where the prevalence of cervical disease was higher. However, a combined use of VIA or VILI with the Pap test or HCII allowed specific detection of cervical abnormalities.

Comparing PAP smear cytology, aided visual inspection, screening colposcopy, cervicography and HPV testing as optional screening tools in Latin America. Study design and baseline data of the LAMS study.

OBJECTIVES: This is a European Commission (EC)-funded ongoing study known as the LAMS (Latin American Screening) study, where PAP smear/liquid-based cytology and screening colposcopy were compared with i) three optional screening tools [visual inspection with acetic acid (VIA), or Lugol's iodine (VILI), cervicography] and with ii) Hybrid Capture II from a) conventional samples and from b) self-samples, in women at different risk for cervical cancer in Brazil and Argentina. STUDY DESIGN: During 2002-2003, a cohort of 12,107 women attending four clinics: Campinas (CA), Sao Paulo (SP), Porto Alegre (PA) and Buenos Aires (BA), were interviewed for risk factors, and examined using the 8 diagnostic arms. Colposcopy was performed for women positive in any test and for 5% of women with baseline PAP-negative and 20% of HCII-negatives. All high-grade lesions (CIN2/3) were treated, and low-grade CIN are prospectively followed-up. RESULTS: Of the 12,107 women, the following baseline data are available: epidemiological data (n=11,996), conventional PAP smears (n=10,363), LBC, SurePATH (n=320), LBC, DNA-Citoliq (n=1,346), VIA (n=12.067), VILI (n=3,061), cervicography (n=279), screening colposcopy (n=3,437), HCII conventional (n=4,710), HCII self-sampling (n=246) and cervical biopsies (n=1,524). The four sub-cohorts differ significantly in all their baseline data on the implicated risk factors of cervical cancer, consonant with their origin from regions with different cancer incidence. Around 95% of all PAP smears were negative, with slight variations in the prevalence of LSIL and HSIL between the four centers. Significant differences were found in the detection rates of abnormal findings in VIA, VILI and colposcopy between the four centers (p=0.0001). The prevalence of HPV was practically identical (16.5-18.8%) in all four cohorts (p=0.486), with no differences in the relative viral loads. Biopsy results were different depending on whether the women underwent screening colposcopy (BA) or elective colposcopy (others). CONCLUSION: Four cohorts with significantly different baseline data are available, and prospective follow-up of these women permits analysis of whether variations in cervical cancer incidence in these regions is due to i) different natural history of the precursor lesions, or ii) due to different levels of exposure to the known risk factors.

Aided visual inspection with acetic acid (VIA) and HPV detection as optional screening tools for cervical cancer and its precursor lesions.

PURPOSE OF INVESTIGATION: To assess the contribution of visual inspection with acetic acid (VIA) and Hybrid Capture II (HCII) as adjunct methods to the Pap test in detecting cervical neoplasia. SUBJECTS AND METHODS: This was a cross-sectional study with 809 women who consecutively attended gynecological consultations at Campinas University, Brazil, from January 2002 to July 2003. Pap test, HCII, VIA, and colposcopy were offered to all patients. Performance of tests (alone or in combination) in detecting histologically confirmed lesions was evaluated. RESULTS: Of the 40 patients with CIN, 69% had CIN1, 26% CIN2 or CIN3 and one patient had invasive carcinoma. VIA had the best performance in detecting CIN, yielding 72% sensitivity and 91% specificity. Considering only CIN2 or worse as significant lesions, HCII had the best sensitivity (73%), while the Pap test was the most specific (93%). Combining the three exams, 92% of the CIN1 or worse were detected. When CIN1 was excluded from the analysis, Pap smear plus HCII delivered 82% sensitivity and 79% specificity. However, this combination yielded a very low (5%) PPV. CONCLUSION: VIA and HCII contributed to the screening of cervical neoplasia in a group of Brazilian women, but the cost-effectiveness of conjoint screening modalities is still debatable.

Intrapreputial infection of young bulls with bovine herpesvirus type 1.2 (BHV-1.2): acute balanoposthitis, latent infection and detection of viral DNA in regional neural and non-neural tissues 50 days after experimental reactivation.

Venereal infection of bulls with bovine herpesvirus type 1.2 (BHV-1.2) may result in acute balanoposthitis followed by the establishment of latent infection, presumably in dorsal root nerve ganglia. We herein report the characterization of the acute and latent infection of young bulls with a Brazilian BHV-1.2 isolate and the investigation of neural and non-neural sites in which viral DNA persists during latent infection, i.e. 110 days after inoculation and 50 days after experimental reactivation. Intrapreputial inoculation of BHV-1.2 isolate SV-56/90 (10(6.5)pfu per animal) resulted in severe balanoposthitis, characterized by redness of the penis and preputial mucosa, coalescent vesicles and fibrinous exsudate in all four infected bulls. Virus shedding was detected in preputial secretions and semen up to days 14 and 13 pi, respectively. Dexamethasone administration at day 60 pi led to reactivation of the infection in all animals, resulting in virus shedding in preputial secretions and/or in semen. At day 50 post-reactivation (pr), the animals were euthanized and regional tissues were collected for PCR and virus isolation. Viral DNA was consistently detected in the dorsal root ganglia of nerves genito-femoral (4/4) and obturator (4/4); frequently in the pudendal (3/4), sciatic (3/4) and rectal caudal nerve ganglia (2/3). In addition, viral DNA was detected in the pelvic sympathetic plexus of one bull and in regional lymph nodes (deep inguinal (2/4); sacral (1/4); medial iliac (1/4)) of two bulls. No infectious virus could be recovered from homogenates of DNA positive tissues, indicating the absence of actively replicating virus. These results demonstrate that BHV-1.2 DNA may persist in several sacral nerve ganglia and in regional lymph nodes as well during latent infection, i.e. 50 days after experimental reactivation. These findings may help in understanding the pathogenesis of acute and latent genital infection by BHV-1.2.

Cardiac malformations, adrenal agenesis, neural crest defects and exencephaly in mice lacking Cited2, a new Tfap2 co-activator.

The protein EP300 and its paralog CREBBP (CREB-binding protein) are ubiquitously expressed transcriptional co-activators and histone acetyl transferases. The gene EP300 is essential for normal cardiac and neural development, whereas CREBBP is essential for neurulation, hematopoietic differentiation, angiogenesis and skeletal and cardiac development. Mutations in CREBBP cause Rubinstein-Taybi syndrome, which is characterized by mental retardation, skeletal abnormalities and congenital cardiac defects. The CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2) binds EP300 and CREBBP with high affinity and regulates gene transcription. Here we show that Cited2-/- embryos die with cardiac malformations, adrenal agenesis, abnormal cranial ganglia and exencephaly. The cardiac defects include atrial and ventricular septal defects, overriding aorta, double-outlet right ventricle, persistent truncus arteriosus and right-sided aortic arches. We find increased apoptosis in the midbrain region and a marked reduction in ErbB3-expressing neural crest cells in mid-embryogenesis. We show that CITED2 interacts with and co-activates all isoforms of transcription factor AP-2 (TFAP2). Transactivation by TFAP2 isoforms is defective in Cited2-/- embryonic fibroblasts and is rescued by ectopically expressed CITED2. As certain Tfap2 isoforms are essential in neural crest, neural tube and cardiac development, we propose that abnormal embryogenesis in mice lacking Cited2 results, at least in part, from its role as a Tfap2 co-activator.

Type I interferon gene expression: differential expression of IFN-A genes induced by viruses and double-stranded RNA.

The family of interferon regulatory transcription factors (IRF) participates in the virus-induced and dsRNA-stimulated transcriptional regulation of either type I IFN genes or a definite set of genes which can also be activated by IFN. In this review, we place emphasis on the role of IRF-3 that associates with the coactivators CBP and/or p300, together or not with IRF-7. These complexes bind to the PRDI, PRDI-like domains or to a number of ISRE sequences located in the promoter of these virus-inducible genes. We also discuss the involvement of the IRF-3-related complexes in the differential regulation of IFN-A genes.

Synergism between multiple virus-induced factor-binding elements involved in the differential expression of interferon A genes.

Comparative transfection analysis of murine interferon A4 and interferon A11 promoter constructs transiently transfected in mouse L929 and human HeLa S3 cells infected with Newcastle disease virus showed that the second positive regulatory domain I-like domain (D motif), located between nucleotides -57 and -46 upstream of the transcription start site, contributes to the activation of virus-induced transcription of the interferon (IFN)-A4 gene promoter by cooperating with the positive regulatory domain I-like and TG-like domains previously described. Electrophoretic mobility shift assay performed with the virus-inducible fragments containing these motifs indicated that the binding activity that we have denoted as virus-induced factor (Génin, P., Bragança, J., Darracq, N., Doly, J., and Civas, A. (1995) Nucleic Acids Res. 23, 5055-5063) is different from interferon-stimulated gene factor 3. It binds to the D motif but not to the virus-unresponsive form of the D motif disrupted by a G-57 --> C substitution. We show that the low levels of IFN-A11 gene expression are caused essentially by the lack of two inducible enhancer domains disrupted by the A-78 --> G and the G-57 --> C substitutions. These data suggest a model taking account of the differential regulation of IFN-A gene family members. They also suggest that virus-induced factor may correspond to the primary transcription factor directly activated by virus that is involved in the initiation of IFN-A gene transcription.

A novel PRD I and TG binding activity involved in virus-induced transcription of IFN-A genes.

Comparative analysis of the inducible elements of the mouse interferon A4 and A11 gene promoters (IE-A4 and IE-A11) by transient transfection experiments, DNase 1 footprinting and electrophoretic mobility shift assays resulted in identification of a virus-induced binding activity suggested to be involved in NDV-induced activation of transcription of these genes. The virus-induced factor, termed VIF, is activated early by contact of virions with cells. It specifically recognizes the PRD I-like domain shared by both inducible elements, as well as the TG-like domain of IE-A4. This factor, distinct from the IRF-1, IRF-2 and the alpha F1 binding proteins and presenting a different affinity pattern from that of the TG protein, is proposed as a candidate for IFN-type I gene regulation.