Differential regulation of human 3β-hydroxysteroid dehydrogenase type 2 for steroid hormone biosynthesis by starvation and cyclic AMP stimulation: studies in the human adrenal NCI-H295R cell model

Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3β-hydroxysteroid dehydrogenases (HSD3Bs). Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute) versus long-term (chronic) stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.

Depressed type 1 cytokine synthesis by superantigen-activated CD4+ T cells of women with human papillomavirus-related high-grade squamous intraepithelial lesions.

Carcinoma of the cervix is causally related to infection with the human papillomavirus (HPV), and T cells play a pivotal role in the immune response of the host to rid itself of HPV infection. Therefore, we assessed the T-cell function of women with HPV-related cervical neoplasia against a superantigen, Staphylococcus enterotoxin B (SEB). Each woman provided a cervical brush specimen for HPV DNA testing and Papanicolaou (Pap) smears for the staging of cervical lesions. They also provided a blood specimen for determination of the ability of CD4(+) T and CD8(+) T cells to synthesize Th1 (interleukin-2 [IL-2], gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and Th2 (IL-10) cytokines in response to activation with SEB. Compared with control subjects with self-attested negative Pap smears, women with high-grade squamous intraepithelial lesions (HSIL) had significantly lower percentages of activated CD4(+) T cells that produced IL-2 (P = 0.045), IFN-gamma (P = 0.040), and TNF-alpha (P = 0.015) and a significantly lower percentage of activated CD8(+) T cells that produced IL-2 (P < 0.01). These data indicate that women with HPV-related cervical HSIL show a decrease in Th1 cytokine production by activated CD4(+) T cells and suggested that compromised T-helper functions may negatively impact the function of cytotoxic CD8(+) T cells.

Maternal and neonatal outcomes by labor onset type and gestational age.

OBJECTIVE: We sought to determine maternal and neonatal outcomes by labor onset type and gestational age. STUDY DESIGN: We used electronic medical records data from 10 US institutions in the Consortium on Safe Labor on 115,528 deliveries from 2002 through 2008. Deliveries were divided by labor onset type (spontaneous, elective induction, indicated induction, unlabored cesarean). Neonatal and maternal outcomes were calculated by labor onset type and gestational age. RESULTS: Neonatal intensive care unit admissions and sepsis improved with each week of gestational age until 39 weeks (P < .001). After adjusting for complications, elective induction of labor was associated with a lower risk of ventilator use (odds ratio [OR], 0.38; 95% confidence interval [CI], 0.28-0.53), sepsis (OR, 0.36; 95% CI, 0.26-0.49), and neonatal intensive care unit admissions (OR, 0.52; 95% CI, 0.48-0.57) compared to spontaneous labor. The relative risk of hysterectomy at term was 3.21 (95% CI, 1.08-9.54) with elective induction, 1.16 (95% CI, 0.24-5.58) with indicated induction, and 6.57 (95% CI, 1.78-24.30) with cesarean without labor compared to spontaneous labor. CONCLUSION: Some neonatal outcomes improved until 39 weeks. Babies born with elective induction are associated with better neonatal outcomes compared to spontaneous labor. Elective induction may be associated with an increased hysterectomy risk.

{\it In vivo\/} induction of cytotoxic T lymphocytes against human immunodeficiency virus type 1 by synthetic viral peptides

Cytotoxic T lymphocytes (CTLs) play an important role in the suppression of initial viremia after acute infection with the human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS). Most HIV-infected individuals attain a high titer of anti-HIV antibodies within weeks of infection; however this antibody-mediated immune response appears not to be protective. In addition, anti-HIV antibodies can be detrimental to the immune response to HIV through enhancement of infection and participating in autoimmune reactions as a result of HIV protein mimicry of self antigens. Thus induction and maintenance of a strong HIV-specific CTL immune response in the absence of anti-HIV antibodies has been proposed to be the most effective means of controlling of HIV infection. Immunization with synthetic peptides representing HIV-specific CTL epitopes provides a way to induce specific CTL responses, while avoiding stimulation of anti-HIV antibody. This dissertation examines the capacity of synthetic peptides from the V3 loop region of the gp120 envelope protein from several different strain of HIV-1 to induce HIV-specific, MHC-restricted CD8$\sp+$ CTL response in vivo in a mouse model. Seven synthetic peptides representative of sequences found throughout North America, Europe, and Central Africa have been shown to prime CTLs in vivo. In the case of the MN strain of HIV-1, a 13 amino acid sequence defining the epitope is most efficient for optimal induction of specific CTL, whereas eight to nine amino acid sequences that could define the epitope were not immunogenic. In addition, synthesis of peptides with specific amino acid substitutions that are important for either MHC binding or T cell receptor recognition resulted in peptides that exhibited increased immunogenicity and induced CTLs that displayed altered specificity. V3 loop peptides from HIV-1 MN, SC, and Z321 induced a CTL population that was broadly cross-reactive against strains of HIV-1 found throughout the world. This research confirms the potential efficacy of using synthetic peptides for in vivo immunization to induce HIV-specific CTL-mediated responses and provides a basis for further research into development of synthetic peptide-based vaccines. ^

Demographic model of the Swiss cattle population for the years 2009-2011 stratified by gender, age and production type

Demographic composition and dynamics of animal and human populations are important determinants for the transmission dynamics of infectious disease and for the effect of infectious disease or environmental disasters on productivity. In many circumstances, demographic data are not available or of poor quality. Since 1999 Switzerland has been recording cattle movements, births, deaths and slaughter in an animal movement database (AMD). The data present in the AMD offers the opportunity for analysing and understanding the dynamic of the Swiss cattle population. A dynamic population model can serve as a building block for future disease transmission models and help policy makers in developing strategies regarding animal health, animal welfare, livestock management and productivity. The Swiss cattle population was therefore modelled using a system of ordinary differential equations. The model was stratified by production type (dairy or beef), age and gender (male and female calves: 0-1 year, heifers and young bulls: 1-2 years, cows and bulls: older than 2 years). The simulation of the Swiss cattle population reflects the observed pattern accurately. Parameters were optimized on the basis of the goodness-of-fit (using the Powell algorithm). The fitted rates were compared with calculated rates from the AMD and differed only marginally. This gives confidence in the fitted rates of parameters that are not directly deductible from the AMD (e.g. the proportion of calves that are moved from the dairy system to fattening plants).

Npro of classical swine fever virus contributes to pathogenicity in pigs by preventing type I interferon induction at local replication sites

Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of pigs. The viral protein Npro of CSFV interferes with alpha- and beta-interferon (IFN-α/β) induction by promoting the degradation of interferon regulatory factor 3 (IRF3). During the establishment of the live attenuated CSF vaccine strain GPE-, Npro acquired a mutation that abolished its capacity to bind and degrade IRF3, rendering it unable to prevent IFN-α/β induction. In a previous study, we showed that the GPE- vaccine virus became pathogenic after forced serial passages in pigs, which was attributed to the amino acid substitutions T830A in the viral proteins E2 and V2475A and A2563V in NS4B. Interestingly, during the re-adaptation of the GPE- vaccine virus in pigs, the IRF3-degrading function of Npro was not recovered. Therefore, we examined whether restoring the ability of Npro to block IFN-α/β induction of both the avirulent and moderately virulent GPE--derived virus would enhance pathogenicity in pigs. Viruses carrying the N136D substitution in Npro regained the ability to degrade IRF3 and suppress IFN-α/β induction in vitro. In pigs, functional Npro significantly reduced the local IFN-α mRNA expression in lymphoid organs while it increased quantities of IFN-α/β in the circulation, and enhanced pathogenicity of the moderately virulent virus. In conclusion, the present study demonstrates that functional Npro influences the innate immune response at local sites of virus replication in pigs and contributes to pathogenicity of CSFV in synergy with viral replication.

Chlorophyll breakdown: Pheophorbide a oxygenase is a Rieske-type iron-sulfur protein, encoded by the accelerated cell death 1 gene

Chlorophyll (chl) breakdown during senescence is an integral part of plant development and leads to the accumulation of colorless catabolites. The loss of green pigment is due to an oxygenolytic opening of the porphyrin macrocycle of pheophorbide (pheide) a followed by a reduction to yield a fluorescent chl catabolite. This step is comprised of the interaction of two enzymes, pheide a oxygenase (PaO) and red chl catabolite reductase. PaO activity is found only during senescence, hence PaO seems to be a key regulator of chl catabolism. Whereas red chl catabolite reductase has been cloned, the nature of PaO has remained elusive. Here we report on the identification of the PaO gene of Arabidopsis thaliana (AtPaO). AtPaO is a Rieske-type iron–sulfur cluster-containing enzyme that is identical to Arabidopsis accelerated cell death 1 and homologous to lethal leaf spot 1 (LLS1) of maize. Biochemical properties of recombinant AtPaO were identical to PaO isolated from a natural source. Production of fluorescent chl catabolite-1 required ferredoxin as an electron source and both substrates, pheide a and molecular oxygen. By using a maize lls1 mutant, the in vivo function of PaO, i.e., degradation of pheide a during senescence, could be confirmed. Thus, lls1 leaves stayed green during dark incubation and accumulated pheide a that caused a light-dependent lesion mimic phenotype. Whereas proteins were degraded similarly in wild type and lls1, a chl-binding protein was selectively retained in the mutant. PaO expression correlated positively with senescence, but the enzyme appeared to be post-translationally regulated as well.

Rotational constants and structure of para-difluorobenzene determined by femtosecond Raman coherence spectroscopy: A new transient type

Femtosecond Raman rotational coherence spectroscopy (RCS) detected by degenerate four-wave mixing is a background-free method that allows to determine accurate gas-phase rotational constants of non-polar molecules. Raman RCS has so far mostly been applied to the regular coherence patterns of symmetric-top molecules, while its application to nonpolar asymmetric tops has been hampered by the large number of RCS transient types, the resulting variability of the RCS patterns, and the 10³–10⁴ times larger computational effort to simulate and fit rotational Raman RCS transients. We present the rotational Raman RCS spectra of the nonpolar asymmetric top 1,4-difluorobenzene (para-difluorobenzene, p-DFB) measured in a pulsed Ar supersonic jet and in a gas cell over delay times up to ~2.5 ns. p-DFB exhibits rotational Raman transitions with ΔJ = 0, 1, 2 and ΔK = 0, 2, leading to the observation of J −, K −, A −, and C–type transients, as well as a novel transient (S–type) that has not been characterized so far. The jet and gas cell RCS measurements were fully analyzed and yield the ground-state (v = 0) rotational constants Aₒ = 5637.68(20) MHz, Bₒ = 1428.23(37) MHz, and Cₒ = 1138.90(48) MHz (1σ uncertainties). Combining the Aₒ, Bₒ, and Cₒ constants with coupled-cluster with single-, double- and perturbatively corrected triple-excitation calculations using large basis sets allows to determine the semi-experimental equilibrium bond lengths rₑ(C₁–C₂) = 1.3849(4) Å, rₑ(C₂–C³) = 1.3917(4) Å, rₑ(C–F) = 1.3422(3) Å, and rₑ(C₂–H₂) = 1.0791(5) Å.

T-cell reprogramming through targeted CD4-coreceptor and T-cell receptor expression on maturing thymocytes by latent Circoviridae family member porcine circovirus type 2 cell infections in the thymus

Although porcine circovirus type 2 (PCV2)-associated diseases have been evaluated for known immune evasion strategies, the pathogenicity of these viruses remained concealed for decades. Surprisingly, the same viruses that cause panzootics in livestock are widespread in young, unaffected animals. Recently, evidence has emerged that circovirus-like viruses are also linked to complex diseases in humans, including children. We detected PCV2 genome-carrying cells in fetal pig thymi. To elucidate virus pathogenicity, we developed a new pig infection model by in vivo transfection of recombinant PCV2 and the immunosuppressant cofactor cyclosporine A. Using flow cytometry, immunofluorescence and fluorescence in situ hybridization, we found evidence that PCV2 dictates positive and negative selection of maturing T cells in the thymus. We show for the first time that PCV2-infected cells reside at the corticomedullary junction of the thymus. In diseased animals, we found polyclonal deletion of single positive cells (SPs) that may result from a loss of major histocompatibility complex class-II expression at the corticomedullary junction. The percentage of PCV2 antigen-presenting cells correlated with the degree of viremia and, in turn, the severity of the defect in thymocyte maturation. Moreover, the reversed T-cell receptor/CD4-coreceptor expression dichotomy on thymocytes at the CD4(+)CD8(interm) and CD4SP cell stage is viremia-dependent, resulting in a specific hypo-responsiveness of T-helper cells. We compare our results with the only other better-studied member of Circoviridae, chicken anemia virus. Our data show that PCV2 infection leads to thymocyte selection dysregulation, adding a valuable dimension to our understanding of virus pathogenicity.

Aortic morphometry at endograft position as assessed by 3D image analysis affects risk of type I endoleak formation after TEVAR

PURPOSE The purpose of this study was to identify morphologic factors affecting type I endoleak formation and bird-beak configuration after thoracic endovascular aortic repair (TEVAR). METHODS Computed tomography (CT) data of 57 patients (40 males; median age, 66 years) undergoing TEVAR for thoracic aortic aneurysm (34 TAA, 19 TAAA) or penetrating aortic ulcer (n = 4) between 2001 and 2010 were retrospectively reviewed. In 28 patients, the Gore TAG® stent-graft was used, followed by the Medtronic Valiant® in 16 cases, the Medtronic Talent® in 8, and the Cook Zenith® in 5 cases. Proximal landing zone (PLZ) was in zone 1 in 13, zone 2 in 13, zone 3 in 23, and zone 4 in 8 patients. In 14 patients (25%), the procedure was urgent or emergent. In each case, pre- and postoperative CT angiography was analyzed using a dedicated image processing workstation and complimentary in-house developed software based on a 3D cylindrical intensity model to calculate aortic arch angulation and conicity of the landing zones (LZ). RESULTS Primary type Ia endoleak rate was 12% (7/57) and subsequent re-intervention rate was 86% (6/7). Left subclavian artery (LSA) coverage (p = 0.036) and conicity of the PLZ (5.9 vs. 2.6 mm; p = 0.016) were significantly associated with an increased type Ia endoleak rate. Bird-beak configuration was observed in 16 patients (28%) and was associated with a smaller radius of the aortic arch curvature (42 vs. 65 mm; p = 0.049). Type Ia endoleak was not associated with a bird-beak configuration (p = 0.388). Primary type Ib endoleak rate was 7% (4/57) and subsequent re-intervention rate was 100%. Conicity of the distal LZ was associated with an increased type Ib endoleak rate (8.3 vs. 2.6 mm; p = 0.038). CONCLUSIONS CT-based 3D aortic morphometry helps to identify risk factors of type I endoleak formation and bird-beak configuration during TEVAR. These factors were LSA coverage and conicity within the landing zones for type I endoleak formation and steep aortic angulation for bird-beak configuration.

Carbohydrate Estimation by a Mobile Phone-Based System Versus Self-Estimations of Individuals With Type 1 Diabetes Mellitus: A Comparative Study

Background: Diabetes mellitus is spreading throughout the world and diabetic individuals have been shown to often assess their food intake inaccurately; therefore, it is a matter of urgency to develop automated diet assessment tools. The recent availability of mobile phones with enhanced capabilities, together with the advances in computer vision, have permitted the development of image analysis apps for the automated assessment of meals. GoCARB is a mobile phone-based system designed to support individuals with type 1 diabetes during daily carbohydrate estimation. In a typical scenario, the user places a reference card next to the dish and acquires two images using a mobile phone. A series of computer vision modules detect the plate and automatically segment and recognize the different food items, while their 3D shape is reconstructed. Finally, the carbohydrate content is calculated by combining the volume of each food item with the nutritional information provided by the USDA Nutrient Database for Standard Reference. Objective: The main objective of this study is to assess the accuracy of the GoCARB prototype when used by individuals with type 1 diabetes and to compare it to their own performance in carbohydrate counting. In addition, the user experience and usability of the system is evaluated by questionnaires. Methods: The study was conducted at the Bern University Hospital, “Inselspital” (Bern, Switzerland) and involved 19 adult volunteers with type 1 diabetes, each participating once. Each study day, a total of six meals of broad diversity were taken from the hospital’s restaurant and presented to the participants. The food items were weighed on a standard balance and the true amount of carbohydrate was calculated from the USDA nutrient database. Participants were asked to count the carbohydrate content of each meal independently and then by using GoCARB. At the end of each session, a questionnaire was completed to assess the user’s experience with GoCARB. Results: The mean absolute error was 27.89 (SD 38.20) grams of carbohydrate for the estimation of participants, whereas the corresponding value for the GoCARB system was 12.28 (SD 9.56) grams of carbohydrate, which was a significantly better performance ( P=.001). In 75.4% (86/114) of the meals, the GoCARB automatic segmentation was successful and 85.1% (291/342) of individual food items were successfully recognized. Most participants found GoCARB easy to use. Conclusions: This study indicates that the system is able to estimate, on average, the carbohydrate content of meals with higher accuracy than individuals with type 1 diabetes can. The participants thought the app was useful and easy to use. GoCARB seems to be a well-accepted supportive mHealth tool for the assessment of served-on-a-plate meals.

Border intervention by Promotores for type 2 diabetes

Purpose of the study. This study had two components. The first component of the study was the development and implementation of an infrastructure that integrated Promotores who teach diabetes self-management into a community clinic. The second component was a six-month randomized clinical trial (RCT) designed to test the effectiveness of the Promotores in changing knowledge, beliefs, and HbA1c levels among Mexican American patients with type 2 diabetes. ^ Methods. Starfield's adaptation of the Donbedian structure, process, and outcome methodology was used to develop a clinic infrastructure that allowed the integration of Promotores as diabetes educators. The RCT of the culturally sensitive Promotores-led 10-week diabetes self-management program compared the outcomes of 63 patients in the intervention group with 68 patients in a wait-list, usual care control group. Participants were Mexican Americans, at least 18 years of age, with type 2 diabetes, who were patients at a Federally Qualified Health Center on the Texas-Mexico border. At baseline, three months, and six months, data were collected using the Diabetes Knowledge Questionnaire (DKQ, the Health Beliefs Questionnaire (HBQ, and HbA1c levels were drawn by the clinic laboratory. A mixed model methodology was used to analyze the data. ^ Results. The infrastructure to support a Promotores-led diabetes self-management course designed in concert with administration, the physicians, and the CDE, resulted in (1) employment of Promotores to teach diabetes self-management courses; (2) integration of provider and nurse oversight of course design and implementation; (3) management of Promotora training, and the development of teaching competencies and skills; (4) coordination of care through communication and documentation policies and procedures; (5) utilization of quality control mechanisms to maintain patient safety; and (6) promotion of a culturally competent approach to the educational process. The RCT resulted in a significant improvement in the intervention group's DKQ scores over time (F [1, 129] = 4.77, p = 0.0308), and in treatment by time (F [2, 168] = 5.85, p = 0.0035). Neither the HBQ scores nor the HbA1c changed over time. However, the baseline HbA1c was 7.49, almost at the therapeutic level. The DKQ, HBQ, and HbA1c results were significantly affected by age; the DKQ and HbA1c by years with diabetes. ^ Conclusions. The clinic model provides a systematic approach to safely address the educational needs of large numbers of patients with type 2 diabetes who live in communities that suffer from a lack of health care professionals. The Promotores-led diabetes self-management course improved the knowledge of patients with diabetes and may be a culturally sensitive strategy for meeting patient educational needs. The low baseline HbA1c levels in this border community suggested that patients in this Federally Qualified Health Center on the Texas-Mexico border were experiencing good medical management of their diabetes. ^