Three-dimensional reconstruction of defects in congenital diaphragmatic hernia: a fetal MRI study.

OBJECTIVE: To assess the clinical feasibility and validity of fetal magnetic resonance imaging (MRI)-based three-dimensional (3D) reconstruction to locate, classify and quantify diaphragmatic defects in congenital diaphragmatic hernia (CDH). METHODS: This retrospective study included 46 cases of CDH which underwent a total of 69 fetal MRI scans (65 in-vivo and four postmortem) at the Medical University of Vienna during the period 1 January 2002 to 1 January 2017. Scans were performed between 16 and 38 gestational weeks using steady-state free precession, T2-weighted and T1-weighted sequences. MRI data were retrieved from the hospital database and manual segmentation of the diaphragm was performed with the open-source software, ITK-SNAP. The resulting 3D models of the fetal diaphragm and its defect(s) were validated by postmortem MRI segmentation and/or comparison of 3D model-based classification of the defect with a reference classification based on autopsy and/or surgery reports. Surface areas of the intact diaphragm and of the defect were measured and used to calculate defect-diaphragmatic ratios (DDR). The need for prosthetic patch repair and, in cases with repeated in-vivo fetal MRI scans, diaphragm growth dynamics, were analyzed based on DDR. RESULTS: Fetal MRI-based manual segmentation of the diaphragm in CDH was feasible for all 65 (100%) of the in-vivo fetal MRI scans. Based on the 3D diaphragmatic models, one bilateral and 45 unilateral defects (n = 47) were further classified as posterolateral (23/47, 48.9%), lateral (7/47, 14.9%) or hemidiaphragmatic (17/47, 36.2%) defects, and none (0%) was classified as anterolateral. This classification of defect location was correct in all 37 (100%) of the cases in which this information could be verified. Nineteen cases had a follow-up fetal MRI scan; in five (26.3%) of these, the initial CDH classification was altered by the results of the second scan. Thirty-three fetuses underwent postnatal diaphragmatic surgical repair; 20 fetuses (all of those with DDR ≥ 54 and 88% of those with DDR > 30) received a diaphragmatic patch, while the other 13 underwent primary surgical repair. Individual DDRs at initial and at follow-up in-vivo fetal MRI correlated significantly (P < 0.001). CONCLUSIONS: MRI-based 3D reconstruction of the fetal diaphragm in CDH has been validated to visualize, locate, classify and quantify the defect. Planning of postnatal surgery may be optimized by MRI-based prediction of the necessity for patch placement and the ability to personalize patch design based on 3D-printable templates. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Methods of applying the 1994 case definition of chronic fatigue syndrome - impact on classification and observed illness characteristics.

BACKGROUND: Multiple case definitions are in use to identify chronic fatigue syndrome (CFS). Even when using the same definition, methods used to apply definitional criteria may affect results. The Centers for Disease Control and Prevention (CDC) conducted two population-based studies estimating CFS prevalence using the 1994 case definition; one relied on direct questions for criteria of fatigue, functional impairment and symptoms (1997 Wichita; Method 1), and the other used subscale score thresholds of standardized questionnaires for criteria (2004 Georgia; Method 2). Compared to previous reports the 2004 CFS prevalence estimate was higher, raising questions about whether changes in the method of operationalizing affected this and illness characteristics. METHODS: The follow-up of the Georgia cohort allowed direct comparison of both methods of applying the 1994 case definition. Of 1961 participants (53 % of eligible) who completed the detailed telephone interview, 919 (47 %) were eligible for and 751 (81 %) underwent clinical evaluation including medical/psychiatric evaluations. Data from the 499 individuals with complete data and without exclusionary conditions was available for this analysis. RESULTS: A total of 86 participants were classified as CFS by one or both methods; 44 cases identified by both methods, 15 only identified by Method 1, and 27 only identified by Method 2 (Kappa 0.63; 95 % confidence interval [CI]: 0.53, 0.73 and concordance 91.59 %). The CFS group identified by both methods were more fatigued, had worse functioning, and more symptoms than those identified by only one method. Moderate to severe depression was noted in only one individual who was classified as CFS by both methods. When comparing the CFS groups identified by only one method, those only identified by Method 2 were either similar to or more severely affected in fatigue, function, and symptoms than those only identified by Method 1. CONCLUSIONS: The two methods demonstrated substantial concordance. While Method 2 classified more participants as CFS, there was no indication that they were less severely ill or more depressed. The classification differences do not fully explain the prevalence increase noted in the 2004 Georgia study. Use of standardized instruments for the major CFS domains provides advantages for disease stratification and comparing CFS patients to other illnesses.

Effects of prey quality and predator body size on prey DNA detection success in a centipede predator.

Predator body size and prey quality are important factors driving prey choice and consumption rates. Both factors might affect prey detection success in PCR-based gut content analysis, potentially resulting in over- or underestimation of feeding rates. Experimental evidence, however, is scarce. We examined how body size and prey quality affect prey DNA detection success in centipede predators. Due to metabolic rates increasing with body size, we hypothesized that prey DNA detection intervals will be shorter in large predators than in smaller ones. Moreover, we hypothesized that prey detection intervals of high-quality prey, defined by low carbon-to-nitrogen ratio will be shorter than in low-quality prey due to faster assimilation. Small, medium and large individuals of centipedes Lithobius spp. (Lithobiidae, Chilopoda) were fed Collembola and allowed to digest prey for up to 168 h post-feeding. To test our second hypothesis, medium-sized lithobiids were fed with either Diptera or Lumbricidae. No significant differences in 50% prey DNA detection success time intervals for a 272-bp prey DNA fragment were found between the predator size groups, indicating that body size does not affect prey DNA detection success. Post-feeding detection intervals were significantly shorter in Lumbricidae and Diptera compared to Collembola prey, apparently supporting the second hypothesis. However, sensitivity of diagnostic PCR differed between prey types, and quantitative PCR revealed that concentration of targeted DNA varied significantly between prey types. This suggests that both DNA concentration and assay sensitivity need to be considered when assessing prey quality effects on prey DNA detection success.

Immunogenicity of quadrivalent human papillomavirus vaccine in organ transplant recipients.

Solid organ transplant recipients are at risk of morbidity from human papillomavirus (HPV)-related diseases. Quadrivalent HPV vaccine is recommended for posttransplant patients but there are no data on vaccine immunogenicity. We determined the immunogenicity of HPV vaccine in a cohort of young adult transplant patients. Patients were immunized with three doses of quadrivalent HPV vaccine containing viral types 6, 11, 16 and 18. Immunogenicity was determined by type-specific viral-like protein ELISA. Four weeks after the last dose of vaccine, a vaccine response was seen in 63.2%, 68.4%, 63.2% and 52.6% for HPV 6, 11, 16 and 18, respectively. Factors that led to reduced immunogenicity were vaccination early after transplant (p = 0.019), having a lung transplant (p = 0.007) and having higher tacrolimus levels (p = 0.048). At 12 months, there were significant declines in antibody titer for all HPV types although the number of patients who remained seropositive did not significantly differ. The vaccine was safe and well tolerated. We show suboptimal immunogenicity of HPV vaccine in transplant patients. This is important for counseling patients who choose to receive this vaccine. Further studies are needed to determine an optimal HPV vaccine type and schedule for this population.

2-Hydroxyestradiol enhances binge onset in female rats and reduces prefrontal cortical dopamine in male rats.

Women are more likely to suffer from a bingeing-related eating disorder, which is surprising, since estradiol reduces meal size and is associated with reduced binge frequency. This apparent contradiction may involve the estradiol metabolite, 2-hydroxyestradiol. We previously reported that female rats had faster escalations in shortening intake during the development of bingeing than did males, but acute administration of 2-hydroxyestradiol increased the intake of vegetable shortening to a greater extent in male rats once bingeing was established. Here, we report two separate studies that follow up these previous findings. In the first, we hypothesized that chronic exposure to 2-hydroxyestradiol would promote escalation of bingeing during binge development in ovariectomized female rats. In the second, we hypothesized that acute exposure to 2-hydroxyestradiol would enhance dopamine signaling in the prefrontal cortex after bingeing was established in male rats. In study 1, non-food-deprived female rats were separated into 3 groups: ovariectomized (OVX) with chronic 2-hydroxyestradiol supplementation (E), OVX with vehicle supplementation (O), and intact with vehicle (I). Each group was given access to an optional source of dietary fat (shortening) on Mon, Wed, and Fri for 4 weeks. 2-hydroxyestradiol supplementation prevented OVX-induced weight gain and enhanced escalation of shortening intake over the four-week period (ps<0.05). Additionally, in week 4, rats in the E group ate significantly more shortening than I controls, less chow than either the O or I group, and had a higher shortening to chow ratio than O or I (ps<0.05). Study 2 indicated that acute injection of 2-hydroxyestradiol abolished shortening-evoked dopamine efflux in the prefrontal cortex of bingeing male rats (p<0.05). Together, these studies indicate that 2-hydroxyestradiol can exacerbate bingeing as it develops and can suppress dopamine signaling in the prefrontal cortex once bingeing is established.

Transtemporal ultrasound application potentially elevates brain temperature: results of an anthropomorphic skull model.

PURPOSE: Transtemporal sonothrombolysis is a tool for a more effective treatment in acute stroke patients. However, some reports revealed side effects, which might be potentially connected to temperature elevation. To gain better insight into cerebral temperature changes during transtemporal sonication, diagnostic and therapeutic ultrasound (US) applications were evaluated using an anthropomorphic skull model. MATERIALS AND METHODS: The impact of diagnostic (PW-Doppler, 1.8-MHz, 0.11 W/cm², TIC 1.2) and therapeutic (1-MHz and 3-MHz, 0.07 - 0.71 W/cm², continuous and pulsed mode) US application on temperature changes was evaluated at the level of muscle/temporal bone (TB), TB/brain, brain and at the middle cerebral artery (MCA) using 4 miniature thermocouples along the US beam. Sonication lasted 120 minutes. RESULTS: Diagnostic ultrasound revealed a maximum temperature increase of 1.45°/0.60°/0.39°/0.41°C (muscle/TB, TB/brain, brain, MCA) after 120 minutes. Therapeutic-1-MHz ultrasound raised temperature by 4.33°/2.02°/1.05 °C/0.81°C (pulsed 1:20) and by 10.38°/4.95°/2.43°/2.08°C (pulsed 1:5) over 120 minutes. Therapeutic-3-MHz US raised temperature by 4.89°/2.56°/1.24/1.25°C (pulsed 1:20) and by 14.77°/6.59°/3.56°/2.86°C (pulsed 1:5) over 120 minutes, respectively. Continuous application of therapeutic US (1-MHz and 3-MHz) led to a temperature increase of 13.86°/3.63°/1.66°/1.48°C and 17.09°/4.28°/1.38/0.99°C within 3 minutes. CONCLUSION: Diagnostic PW-Doppler showed only a moderate temperature increase and can be considered as safe. Therapeutic sonication is very powerful in delivering energy so that even pulsed application modes resulted in significant and potentially harmful temperature increases.

Thermal effects of diagnostic ultrasound in an anthropomorphic skull model.

PURPOSE: Exposure to diagnostic ultrasound (US) can significantly heat biological tissue although conventional routine examinations are regarded as safe. The risk of unwanted thermal effects increases with a high absorption coefficient and extended insonation time. Certain applications of transcranial diagnostic US (TC-US) require prolonged exposure. An anthropomorphic skull model (ASM) was developed to evaluate thermal effects induced by TC-US of different modalities. The objective was to determine whether prolonged continuous TC-US application results in potentially harmful temperature increases. MATERIALS AND METHODS: The ASM consists of a human skull with tissue mimicking material and exhibits acoustic and anatomical characteristics of the human skull and brain. Experiments are performed with a diagnostic US device testing four different US modalities: Duplex PW (pulsed wave) Doppler, PW Doppler, color flow Doppler and B-mode. Temperature changes are recorded during 180 minutes of insonation. RESULTS: All measurements revealed significant temperature increases during insonation independent of the US modality. The maximum temperature elevation of + 5.25° C (p < 0.001) was observed on the surface of the skull exposed to duplex PW Doppler. At the bone-brain border a maximum temperature increae of + 2.01 °C (p < 0.001) was noted. Temperature increases within the brain were < 1.23 °C (p = 0.001). The highest values were registered using the duplex PW Doppler modality. CONCLUSION: TC-US induces significant local heating effects in an ASM. An application duration that extends routine clinical periods causes potentially harmful heating especially in tissue close to bone. TC-US elevates the temperature in the brain mimicking tissue but is not capable of producing harmful temperature increases during routine examinations. However, the risk of thermal injury in brain tissue increases significantly after an exposure time of > 2 hours.

Role of solution concentration in formation kinetics of bromide perovskite thin films during spin-coating monitored by optical in situ metrology.

Optoelectronic devices based on metal halide perovskites continue to show a improved performance, and solution-based coating techniques pave the way for large-area applications. However, not all parameters influencing the thin film formation process of metal halide perovskites are identified and entirely rationalised over their full compositional range, thus hampering optimised thin film fabrication. Furthermore, while the perovskite deposition via spin-coating and annealing is an easily accessible technique, more profound insights into the chemical formation process are still lacking. Varying the precursor solution concentration is commonly used to vary the resulting thin film thickness. This study shows that varying the precursor solution concentration also affects the thin film morphology and optoelectronic quality. Hence, we herein investigate the influence of the precursor solution concentration on the formation process of a pure bromide-based triple cation perovskite (Cs0.05MA0.10FA0.85PbBr3) by fiber-based optical in situ measurement. During the spin-coating process, in situ UV-vis and PL measurements reveal formation kinetics are strongly dependent on the concentration. Furthermore, we identify delayed nucleation and retarded growth kinetics for more concentrated precursor solutions. In addition, we quantify the shifting chemical equilibrium of colloidal pre-coordination in the precursor solution depending on concentration. Namely, colloids are pre-organised to a higher degree and higher-coordination lead-bromide complexes tend to form in more concentrated precursor solutions. Thus, the modified solution chemistry rationalises retarded perovskite formation kinetics and highlights the precursor concentration as an influential and optimisable parameter for solution-based thin film deposition.

Monitoring of human papillomavirus vaccination.

Persistent infection with oncogenic human papillomavirus (HPV) is a necessary causal factor in the development of cervical cancer. Moreover, HPV, predominately type 16 and to a lesser degree type 18, is linked causally to varying proportions of other anogenital cancers (vulva, vagina, penis, anus) as well as cancers elsewhere in the body (oropharynx, larynx, conjunctiva). HPV types 6 and 11 cause most of genital warts and recurrent respiratory papillomatosis. Effective prophylactic vaccines have been developed. In this review, we address briefly the immunological aspects of HPV infection and the results of HPV vaccination trials. Internationally standardized monitoring and evaluation of prophylactic HPV vaccination programmes will be essential for arriving at the most cost-effective strategies for cancer control.

Factors associated with prevalent abnormal anal cytology in a large cohort of HIV-infected adults in the United States.

BACKGROUND: The prevalence of and risk factors for abnormal anal cytology among men and women with human immunodeficiency virus (HIV) infection have not been extensively investigated. METHODS: The Study to Understand the Natural History of HIV and AIDS in the Era of Effective Therapy (SUN study) is a prospective cohort study of HIV-infected patients in 4 US cities. Baseline questionnaires were administered and anal samples for cytology and human papillomavirus (HPV) detection and genotyping were collected. RESULTS: Among 471 men and 150 women (median age, 41 years), 78% of participants were receiving combination antiretroviral therapy, 41% had a CD4(+) cell count of ≥500 cells/µL, and 71% had an HIV RNA viral load of <400 copies/mL. The anal cytology results were as follows: 336 participants (54%) had negative results, 96 participants (15%) had atypical squamous cells, 149 participants (24%) had low-grade squamous intraepithelial lesions, and 40 participants (6%) had high-grade squamous intraepithelial lesions. In a multivariate analysis, abnormal anal cytology was associated with number of high-risk and low-risk HPV types (adjusted odds ratio [AOR] for both, 1.28; P < .001), nadir CD4(+) cell count of <50 cells/µL (AOR, 2.38; P = .001), baseline CD4(+) cell count of <500 cells/µL (AOR, 1.75; P = .004), and ever having receptive anal intercourse (AOR, 2.51; P < .001). CONCLUSION: HIV-infected persons with multiple anal HPV types or a nadir CD4(+) cell count of <50 cells/µL have an increased risk for abnormal anal cytology.

Multiplex immunoassay to measure antibody response to nine HPV vaccine types.

Well-characterized HPV serology assays are required to evaluate performance of biosimilar candidate vaccines, reduced dosing schedules and novel administration methods. We report characterization of an expanded assay, M9ELISA, that detects antibodies to HPV virus-like particles (VLP) of nine types using direct IgG ELISA on the Meso Scale Discovery (MSD) electrochemiluminescence platform. The method is based on the previously published M4ELISA which detects antibodies to HPV6,11,16, and 18. It has been modified to add detection of antibodies to HPV31,33,45,52 and 58, and to streamline assay and reduce background. The M9ELISA plates were prepared with purified type specific L1 + L2 VLPs coated on 10-spot/well standard MSD microplates. Results of ELISA on three serial dilutions of serum were read on MSD imager, and titers calculated using the parallel line method. Evaluations included dynamic range, assay reproducibility, and stability over time. We compared M9ELISA results to those from a pseudovirion-based neutralization assay in sera from a mixed cohort of unvaccinated and vaccinated individuals (n = ~116) and to competitive Luminex immunoassay (cLIA) results in sera from a predominantly unvaccinated cohort (n = 4426). The linear range of the assay extended over 5 logs, with inter-assay reproducibility coefficient of variation ≤25% for all types. The pre-coated plates were stable for at least 2 years. Spearman correlation of antibody titers showed excellent correlation with PBNA (r = 0.86-0.97) and moderate correlation (r = 0.52-0.68) with cLIA. Thus, the M9ELISA can serve as a useful platform for high-throughput, sensitive and simultaneous quantitation of the antibody responses to nine HPV vaccine types.

Human papillomavirus (HPV) types among Alaska native women attending a colposcopy clinic in Anchorage, Alaska, 2009-2011.

BACKGROUND: The first HPV vaccines licensed targeted two HPV types responsible for most cervical cancers. A 9-valent vaccine (9vHPV), targeting 5 additional types, was introduced in 2016 and is currently the only HPV vaccine available in the United States. Previous studies demonstrated high rates of HPV infection in Alaska Native (AN) women. We sought to measure prevalence of high risk HPV types in AN women undergoing colposcopy and to determine those preventable by vaccination. METHODS: For this cross-sectional study, we recruited women who were undergoing colposcopy for clinical indications at Alaska Native Medical Center to obtain cervical brush biopsy samples. Specimens were shipped to Atlanta, Georgia for DNA extraction, HPV detection, and typing using L1 PCR with type-specific hybridization to detect 37 HPV types. RESULTS: Four hundred eighty eight specimens from 489 women were tested. At least one HPV type was found in 458 (94%) specimens. Of 458 participants who were HPV positive, 332 (72%) had two or more types. At least one type targeted by 9vHPV was detected in 95% of participants with CIN 3 (21/22), 82% with CIN 2 (37/45), and 65% with CIN 1 (119/184). (p < 0.001) HPV 16 or 18 were detected in 77% (17/22) with CIN 3, 53% (24/45) with CIN 2, and 36% (67/184) with CIN 1. (p < 0.001). CONCLUSIONS: A substantial proportion of AN women attending colposcopy clinic had evidence of HPV 16/18 infection, as well as other high risk types targeted by 9vHPV. At least one 9vHPV type was detected in 62% of the participants overall, and 95% of participants with CIN3. AN women are expected to benefit from vaccination against HPV 16/18, and will have greater benefit from 9vHPV. Information from this study could be used to develop public health strategies to increase vaccine uptake, or to track HPV genotype prevalence over time.

Location of actin, myosin, and microtubular structures during directed locomotion of Dictyostelium amebae.

During their life cycle, amebae of the cellular slime mould Dictyostelium discoideum aggregate to form multicellular structures in which differentiation takes place. Aggregation depends upon the release of chemotactic signals of 3',5'-cAMP from aggregation centers. In response to the signals, aggregating amebae elongate, actively more toward the attractive source, and may be easily identified from the other cells because of their polarized appearance. To examine the role of cytoskeletal components during ameboid locomotion, immunofluorescence microscopy with antibodies to actin, myosin, and to a microtubule-associated component was used. In addition, rhodamine-labeled phallotoxin was employed. Actin and myosin display a rather uniform distribution in rounded unstretched cells. In polarized locomoting cells, actin fluorescence (due to both labeled phallotoxin and specific antibody) is prevalently concentrated in the anterior pseudopod while myosin fluorescence appears to be excluded from the pseudopod. Similarly, microtubules in locomoting cells are excluded from the leading pseudopod. The cell nucleus is attached to the microtubule network by way of a nucleus-associated organelle serving as a microtubule-organizing center and seems to be maintained in a rather fixed position by the microtubules. These findings, together with available morphological and biochemical evidences, are consistent with a mechanism in which polymerized actin is moved into the pseudopod through its interaction with myosin at the base of the pseudopod. Microtubules, apparently, do not actively participate in movement but seem to behave as anchorage structures for the nucleus and possibly other cytoplasmic organelles.

Heparan sulfate proteoglycans are concentrated on the sinusoidal plasmalemmal domain and in intracellular organelles of hepatocytes.

The distribution of cell surface heparan sulfate proteoglycans (HSPGs) was determined in rat liver by immunocytochemistry. A polyclonal antibody was raised against HSPGs purified from rat liver microsomes which specifically immunoprecipitated liver membrane HSPGs. It was shown to recognize both the heparin-releasable and membrane-intercalated form of membrane HSPGs and to recognize determinants on the core protein of these HSPGs. By immunocytochemistry membrane HSPGs were localized to hepatocytes. The distribution of HSPGs at the cell surface of the hepatocyte was restricted to the sinusoidal domain of the plasmalemma; there was little or no staining of the lateral or bile canalicular domains. Intracellularly, HSPGs were occasionally detected in cisternae of the rough endoplasmic reticulum and were regularly found in Golgi cisternae--usually distributed across the entire Golgi stack. HSPGs were also localized in some endosomes, lysosomes, and cytoplasmic vesicles of hepatocytes. We conclude that the HSPGs recognized by this antibody have a restricted distribution in rat liver: they are largely confined to the sinusoidal plasmalemmal domain and to biosynthetic and endocytic compartments of hepatocytes.

Seroprevalence of HPV 6, 11, 16 and 18 and correlates of exposure in unvaccinated women aged 16-64 years in Puerto Rico.

BACKGROUND: To understand risk factors for HPV exposure in Puerto Rican women, we evaluated HPV 6, 11, 16, and 18 serology in women aged living in the San Juan metropolitan area. METHODS: As part of a cross-sectional study, a population-based sample of 524 HPV unvaccinated Hispanic women ages 16-64 years completed face-to-face and computer assisted interviews and provided blood and self-collected anal and cervical specimens. Serology used multiplex virus-like particle based-IgG ELISA and HPV DNA was detected with L1-consensus PCR. RESULTS: 32% and 47% were seropositive to HPV types included in the bivalent (16/18) and quadrivalent (6/11/16/18) vaccines, respectively. Type-specific seroprevalence was HPV6 - 29%, HPV11 - 18%, HPV16 - 23%, and HPV18 - 17%; seroprevalence was high in the youngest age-group (16-19: 26-37%). HPV seropositivity was associated with having ≥ 3 lifetime sexual partners (OR=2.5, 95% CI=1.7-3.9) and detection of anogenital HPV DNA (OR=1.8, 95% CI=1.2-2.6). CONCLUSIONS: The high cumulative exposure of HPV vaccine types 6/11/16/18 in this Hispanic population was influenced by factors related to HPV exposure through sexual behavior. High seroprevalence in the youngest age-group indicates early age of exposure to HPV in Puerto Rico, highlighting the need for HPV vaccination starting prior to age 16.

Glucocorticoid receptor polymorphisms and haplotypes associated with chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a significant public health problem of unknown etiology, the pathophysiology has not been elucidated, and there are no characteristic physical signs or laboratory abnormalities. Some studies have indicated an association of CFS with deregulation of immune functions and hypothalamic-pituitary-adrenal (HPA) axis activity. In this study, we examined the association of sequence variations in the glucocorticoid receptor gene (NR3C1) with CFS because NR3C1 is a major effector of the HPA axis. There were 137 study participants (40 with CFS, 55 with insufficient symptoms or fatigue, termed as ISF, and 42 non-fatigued controls) who were clinically evaluated and identified from the general population of Wichita, KS. Nine single nucleotide polymorphisms (SNPs) in NR3C1 were tested for association of polymorphisms and haplotypes with CFS. We observed an association of multiple SNPs with chronic fatigue compared to non-fatigued (NF) subjects (P < 0.05) and found similar associations with quantitative assessments of functional impairment (by the SF-36), with fatigue (by the Multidimensional Fatigue Inventory) and with symptoms (assessed by the Centers for Disease Control Symptom Inventory). Subjects homozygous for the major allele of all associated SNPs were at increased risk for CFS with odds ratios ranging from 2.61 (CI 1.05-6.45) to 3.00 (CI 1.12-8.05). Five SNPs, covering a region of approximately 80 kb, demonstrated high linkage disequilibrium (LD) in CFS, but LD gradually declined in ISF to NF subjects. Furthermore, haplotype analysis of the region in LD identified two associated haplotypes with opposite alleles: one protective and the other conferring risk of CFS. These results demonstrate NR3C1 as a potential mediator of chronic fatigue, and implicate variations in the 5' region of NR3C1 as a possible mechanism through which the alterations in HPA axis regulation and behavioural characteristics of CFS may manifest.

Dominant Negative Mutants of Opaque2 Suppress Transactivation of a 22-kD Zein Promoter by Opaque2 in Maize Endosperm Cells.

In maize endosperm, genes encoding the 22-kD zein class of storage proteins are regulated by the OPAQUE2 locus. The Opaque2 (O2) protein shares homology with the basic domain/leucine zipper class of transcriptional activators. Using microprojectile bombardment, we have shown that O2 is capable of transactivating a 22-kD zein promoter in maize endosperm suspension cultures and in longitudinal sections of intact endosperm. Two mutant forms of the O2 gene were constructed by deleting regions that encode either the basic domain or the first 175 N-terminal residues of the O2 protein. When either of these mutant O2 genes was coexpressed with wild-type O2 in a maize endosperm expression system, O2-mediated transactivation of the 22-kD zein promoter was inhibited specifically and in a dose-dependent manner. Electrophoretic mobility shift assays and immunoprecipitation studies indicated that the mutant O2 proteins form heterodimers with wild-type O2 in vitro. The mutant lacking the basic domain forms heterodimers with wild-type O2, which can no longer bind DNA. In contrast, the product of the N-terminal truncation allele forms homodimers and heterodimers with wild-type O2, both of which can still bind DNA. Because the N-terminal region contains an activation domain, it is likely that these latter complexes are deficient in transactivation. Dominant negative inhibitors of gene expression, such as those constructed here, provide an alternative to antisense RNA approaches for inactivation of gene function in plants.

In vitro induction of cell-mediated immunity to murine leukemia cells. III. Effect of methanol extraction residue fraction of BCG on the generation of cytotoxic lymphocytes against leukemia.

Effects of the methanol extraction residue (MER) fraction of tubercle bacilli (BCG) on the generation of cytotoxic lymphoid cells were studied in vitro with the use of unidirectional mixed lymphocyte-tumor cell cultures. These cultures consisted of splenocytes of lymph node cells from normal donor C57BL/6, BALB/c, and strain A mice and mitomycin C-inactivated leukemia cells of both syngeneic and allogeneic origin. Addition of small amounts of MER (0.2-5 microgram/ml) to the cultures potentiated appreciably the elicitation of cytotoxic reactivity (as measured by the 51Cr-release assay) of the sensitized cells, whereas higher quantities (10-40 microgram/ml) had a strong suppressive effect. MER also induced some cytotoxic capacity in normal murine and human lymphoid cells not exposed to specific tumor cell stimulation. The stimulatory and suppressive effects were noted only when MER was present during the initial 24-48 hours of the 6-day culture. With the nylon wool fractionation technique, it was apparent that MER affected primarily the nonadherent cell population. MER could also prevent the generation of nonspecific suppressor cells by splenocytes maintained for 3-6 days in tissue culture.