Huntington’s disease (HD) is a neurodegenerative disorder characterized by chorea,

behavioral disturbances and dementia, caused by a pathological expansion of the CAG

trinucleotide in the HTT gene. Several patients have been recognized with the typical HD

phenotype without the expected mutation. The objective of this study was to assess the

occurrence of diseases such as Huntington’s disease-like 2 (HDL2), spinocerebellar ataxia

(SCA) 1, SCA2, SCA3, SCA7, dentatorubral-pallidoluysian atrophy (DRPLA) and choreaacanthocytosis

(ChAc) among 29 Brazilian patients with a HD-like phenotype. In the group

analyzed, we found 3 patients with HDL2 and 2 patients with ChAc. The diagnosis was not

reached in 79.3% of the patients. HDL2 was the main cause of the HD-like phenotype in

the group analyzed, and is attributable to the African ancestry of this population. However,

the etiology of the disease remains undetermined in the majority of the HD negative

patients with HD-like phenotype.

Key words: Huntington’s disease, Huntington’s disease-like, chorea-acanthocytosis,

Huntington’s disease-like 2.

Background: We analyzed prospectively whether MGMT (O(6)-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression.

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Abstract

Background

We analyzed prospectively whether MGMT (O6-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression.

Methodology/Principal Findings:

Adult patients with a histologically proven malignant astrocytoma (glioblastoma: N = 53, anaplastic astrocytoma: N = 10) were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP) and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA) database (N = 229 glioblastomas). Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001); the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001); however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6) did significantly worse than those with low transcriptional activity (p<0.01). Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6) did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001). Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT mRNA expression.

Conclusions/Significance:

MGMT mRNA expression plays a direct role for mediating tumor sensitivity to alkylating agents. Discordant findings indicate methylation-independent pathways of MGMT expression regulation. DNMT1 and DNMT3b are likely to be involved in CGI methylation. However, their exact role yet has to be defined.

Background: The fetal immune system is characterized by a Th2 bias but it is unclear how the Th2 predominance is established. Natural killer T (NKT) cells are a rare subset of T cells with immune regulatory functions and are already activated in utero. To test the hypothesis that NKT cells are part of the regulatory network that sets the fetal Th2 predominance, percentages of Vα24(+)Vβ11(+) NKT cells expressing Th1/Th2-related chemokine receptors (CKR) were assessed in cord blood. Furthermore, IL-4 and IFN-γ secreting NKT cells were quantified within the single CKR(+) subsets.

Results:

Cord blood NKT cells expressed the Th2-related CCR4 and CCR8 at significantly higher frequencies compared to peripheral blood NKT cells from adults, while CXCR3+ and CCR5+ cord blood NKT cells (Th1-related) were present at lower percentages. Within CD4negCD8neg (DN) NKT cells, the frequency of IL-4 producing NKT cells was significantly higher in cord blood, while frequencies of IFN-γ secreting DN NKT cells tended to be lower. A further subanalysis showed that the higher percentage of IL-4 secreting DN NKT cells was restricted to CCR3+, CCR4+, CCR5+, CCR6+, CCR7+, CCR8+ and CXCR4+ DN subsets in cord blood. This resulted in significantly decreased IFN-γ /IL-4 ratios of CCR3+, CCR6+ and CCR8+ cord blood DN NKT cells. Sequencing of VA24AJ18 T cell receptor (TCR) transcripts in sorted cord blood Vα24Vβ11 cells confirmed the invariant TCR alpha-chain ruling out the possibility that these cells represent an unusual subset of conventional T cells.

Conclusions:

Despite the heterogeneity of cord blood NKT cells, we observed a clear Th2-bias at the phenotypic and functional level which was mainly found in the DN subset. Therefore, we speculate that NKT cells are important for the initiation and control of the fetal Th2 environment which is needed to maintain tolerance towards self-antigens as well as non-inherited maternal antigens.

Background: We recently showed that in preschoolers risk factors for overweight show stronger associations with BMI in children with high BMI values. However, it is unclear whether these findings might also pertain to adolescents.

Methods:

We extracted data on 3–10 year-old (n = 7,237) and 11–17 year-old (n = 5,986) children from a representative cross-sectional German health survey (KiGGS) conducted between 2003 and 2006 and calculated quantile regression models for each age group. We used z-scores of children's body mass index (BMI) as outcome variable and maternal BMI, maternal smoking in pregnancy, low parental socioeconomic status, exclusive formula-feeding and high TV viewing time as explanatory variables.

Results:

In both age groups, the estimated effects of all risk factors except formula-feeding on BMI z-score were greatest for children with the highest BMI z-score. The median BMI z-score of 11–17 year-old children with high TV viewing time, for example, was 0.11 [95% CI: 0.03, 0.19] units higher than the median BMI z-score of teenage children with low TV viewing time. This risk factor was associated with an average difference of 0.18 [0.06, 0.30] units at the 90th percentile of BMI z-score and of 0.20 [0.07, 0.33] units at the 97th percentile.

Conclusions:

We confirmed that risk factors for childhood overweight are associated with greater shifts in the upper parts of the children's BMI distribution than in the middle and lower parts. These findings pertain also to teenagers and might possibly help to explain the secular shift in the upper BMI percentiles in children and adolescents.

Background: A crucial impediment to global tuberculosis control is the lack of an accurate, rapid diagnostic test for detection of patients with active TB. A new, rapid diagnostic method, (Cepheid) Xpert MTB/RIF Assay, is an automated sample preparation and real-time PCR instrument, which was shown to have good potential as an alternative to current reference standard sputum microscopy and culture.

Methods: We performed a clinical validation study on diagnostic accuracy of the Xpert MTB/RIF Assay in a TB and HIV endemic setting. Sputum samples from 292 consecutively enrolled adults from Mbeya, Tanzania, with suspected TB were subject to analysis by the Xpert MTB/RIF Assay. The diagnostic performance of Xpert MTB/RIF Assay was compared to standard sputum smear microscopy and culture. Confirmed Mycobacterium tuberculosis in a positive culture was used as a reference standard for TB diagnosis.

Results: Xpert MTB/RIF Assay achieved 88.4% (95%CI = 78.4% to 94.9%) sensitivity among patients with a positive culture and 99% (95%CI = 94.7% to 100.0%) specificity in patients who had no TB. HIV status did not affect test performance in 172 HIV-infected patients (58.9% of all participants). Seven additional cases (9.1% of 77) were detected by Xpert MTB/RIF Assay among the group of patients with clinical TB who were culture negative. Within 45 sputum samples which grew non-tuberculous mycobacteria the assay's specificity was 97.8% (95%CI = 88.2% to 99.9%).

Conclusions: The Xpert MTB/RIF Assay is a highly sensitive, specific and rapid method for diagnosing TB which has potential to complement the current reference standard of TB diagnostics and increase its overall sensitivity. Its usefulness in detecting sputum smear and culture negative patients needs further study. Further evaluation in high burden TB and HIV areas under programmatic health care settings to ascertain applicability, cost-effectiveness, robustness and local acceptance are required.

Background: Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches blood perfusion to alveolar ventilation to optimize gas exchange. Recently we have demonstrated that acute but not sustained HPV is critically dependent on the classical transient receptor potential 6 (TRPC6) channel. However, the mechanism of TRPC6 activation during acute HPV remains elusive. We hypothesize that a diacylglycerol (DAG)-dependent activation of TRPC6 regulates acute HPV.

Methods:

We investigated the effect of the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) on normoxic vascular tone in isolated perfused and ventilated mouse lungs from TRPC6-deficient and wild-type mice. Moreover, the effects of OAG, the DAG kinase inhibitor R59949 and the phospholipase C inhibitor U73122 on the strength of HPV were investigated compared to those on non-hypoxia-induced vasoconstriction elicited by the thromboxane mimeticum U46619.

Results:

OAG increased normoxic vascular tone in lungs from wild-type mice, but not in lungs from TRPC6-deficient mice. Under conditions of repetitive hypoxic ventilation, OAG as well as R59949 dose-dependently attenuated the strength of acute HPV whereas U46619-induced vasoconstrictions were not reduced. Like OAG, R59949 mimicked HPV, since it induced a dose-dependent vasoconstriction during normoxic ventilation. In contrast, U73122, a blocker of DAG synthesis, inhibited acute HPV whereas U73343, the inactive form of U73122, had no effect on HPV.

Conclusion:

These findings support the conclusion that the TRPC6-dependency of acute HPV is induced via DAG.

Background: Although stroke is acknowledged as a long-term condition, population estimates of outcomes longer term are lacking. Such estimates would be useful for planning health services and developing research that might ultimately improve outcomes. This burden of disease study provides population-based estimates of outcomes with a focus on disability, cognition, and psychological outcomes up to 10 y after initial stroke event in a multi-ethnic European population. Methods and Findings: Data were collected from the population-based South London Stroke Register, a prospective population-based register documenting all first in a lifetime strokes since 1 January 1995 in a multi-ethnic inner city population. The outcomes assessed are reported as estimates of need and included disability (Barthel Index <15), inactivity (Frenchay Activities Index <15), cognitive impairment (Abbreviated Mental Test < 8 or Mini-Mental State Exam <24), anxiety and depression (Hospital Anxiety and Depression Scale > 10), and mental and physical domain scores of the Medical Outcomes Study 12-item short form (SF-12) health survey. Estimates were stratified by age, gender, and ethnicity, and age-adjusted using the standard European population. Plots of outcome estimates over time were constructed to examine temporal trends and sociodemographic differences. Between 1995 and 2006, 3,373 first-ever strokes were registered: 20%-30% of survivors had a poor outcome over 10 y of follow-up. The highest rate of disability was observed 7 d after stroke and remained at around 110 per 1,000 stroke survivors from 3 mo to 10 y. Rates of inactivity and cognitive impairment both declined up to 1 y (280/1,000 and 180/1,000 survivors, respectively); thereafter rates of inactivity remained stable till year eight, then increased, whereas rates of cognitive impairment fluctuated till year eight, then increased. Anxiety and depression showed some fluctuation over time, with a rate of 350 and 310 per 1,000 stroke survivors, respectively. SF-12 scores showed little variation from 3 mo to 10 y after stroke. Inactivity was higher in males at all time points, and in white compared to black stroke survivors, although black survivors reported better outcomes in the SF-12 physical domain. No other major differences were observed by gender or ethnicity. Increased age was associated with higher rates of disability, inactivity, and cognitive impairment. Conclusions: Between 20% and 30% of stroke survivors have a poor range of outcomes up to 10 y after stroke. Such epidemiological data demonstrate the sociodemographic groups that are most affected longer term and should be used to develop longer term management strategies that reduce the significant poor outcomes of this group, for whom effective interventions are currently elusive.

Multiple sclerosis (MS) has a great impact on functioning and disability. The perspective of those who experience the health problem has to be taken into account to obtain an in-depth understanding of functioning and disability. The objective was to describe the areas of functioning and disability and relevant contextual factors in MS from the patient perspective. A qualitative study using focus group methodology was performed. The sample size was determined by saturation. The focus groups were digitally recorded and transcribed verbatim. The meaning condensation procedure was used for data analysis. Identified concepts were linked to International Classification of Functioning, Disability and Health (ICF) categories according to established linking rules. Six focus groups with a total of 27 participants were performed. In total, 1327 concepts were identified and linked to 106 ICF categories of the ICF components Body Functions, Activities and Participation and Environmental Factors. This qualitative study reports on the impact of MS on functioning and disability from the patient perspective. The participants in this study provided information about all physical aspects and areas of daily life affected by the disease, as well as the environmental factors influencing their lives.

Second and Third Harmonic Generation (SHG and THG) microscopy is based on optical effects which are induced by specific inherent physical properties of a specimen. As a multi-photon laser scanning approach which is not based on fluorescence it combines the advantages of a label-free technique with restriction of signal generation to the focal plane, thus allowing high resolution 3D reconstruction of image volumes without out-of-focus background several hundred micrometers deep into the tissue. While in mammalian soft tissues SHG is mostly restricted to collagen fibers and striated muscle myosin, THG is induced at a large variety of structures, since it is generated at interfaces such as refraction index changes within the focal volume of the excitation laser. Besides, colorants such as hemoglobin can cause resonance enhancement, leading to intense THG signals. We applied SHG and THG microscopy to murine (Mus musculus) muscles, an established model system for physiological research, to investigate their potential for label-free tissue imaging. In addition to collagen fibers and muscle fiber substructure, THG allowed us to visualize blood vessel walls and erythrocytes as well as white blood cells adhering to vessel walls, residing in or moving through the extravascular tissue. Moreover peripheral nerve fibers could be clearly identified. Structure down to the nuclear chromatin distribution was visualized in 3D and with more detail than obtainable by bright field microscopy. To our knowledge, most of these objects have not been visualized previously by THG or any label-free 3D approach. THG allows label-free microscopy with inherent optical sectioning and therefore may offer similar improvements compared to bright field microscopy as does confocal laser scanning microscopy compared to conventional fluorescence microscopy.